Expression of the CXCL12/CXCR4 and CXCL16/CXCR6 axes incervical in traepithelial neoplasia and cervical cancer

The chemokine CXCL12 is highly expressed in gynecologic tumors and is widely known to play a biologically relevant role in tumor growth and spread. Recent evidence suggests that CXCL16, a novel chemokine, is overexpressed in inflammation-associated tumors and mediates pro-tumorigenic effects of inflammation in prostate cancer. We therefore analyzed the expression of CXCL12 and CXCL16 and their respective receptors CXCR4 and CXCR6 in cervical intraepithelial neoplasia (CIN) and cervical cancer and further assessed their association with clinicopathologic features and outcomes. Tissue chip technology and immunohistochemistry were used to analyze the expression of CXCL12, CXCR4, CXCL16, and CXCR6 in healthy cervical tissue (21 cases), CIN (65 cases), and cervical carcinoma (60 cases). The association of protein expression with clinicopathologic features and overall survival was analyzed. These four proteins were clearly detected in membrane and cytoplasm of neoplastic epithelial cells, and their distribution and intensity of expression increased as neoplastic lesions progressed through CIN1, CIN2, and CIN3 to invasive cancer. Furthermore, the expression of CXCR4 was associated significantly with the histologic grade of cervical carcinoma, whereas the expression of CXCR6 was associated significantly with lymph node metastasis. In Kaplan-Meier analysis, patients with high CXCR6 expression had significantly shorter overall survival than did those with low CXCR6 expression. The elevated co-expression levels of CXCL12/CXCR4 and CXCL16/CXCR6 in CIN and cervical carcinoma suggest a durative process in cervical carcinoma development. Moreover, CXCR6 may be useful as a biomarker and a valuable prognostic factor for cervical cancer.

Impact of the number of examined lymph nodes on outcomes in patients with lymph node-negative gallbladder carcinoma

AIM To determine whether the number of examined lymph nodes(LNs) is correlated with the overall survival of gallbladder carcinoma(GBC) patients. METHODS Patients were collected from the Surveillance Epidemiology and End Results database(2004-2013) and categorized by the number of LNs into six groups: 1 LN, 2 LNs, 3 LNs, 4 LNs, 5 LNs, and ≥ 6 LNs. Survival curves for overall survival were plotted with a KaplanMeier analysis. The log-rank test was used for univariate comparisons.RESULTS In a cohort of 893 patients, the median number of examined LNs was two for the entire cohort. The survival for the 1 LN group was significantly poorer than those of the stage Ⅰ and Ⅱ disease groups and for the entire cohort. By dichotomizing the number of LNs from 1 to 6, we found that the minimum number of LNs that should be examined was four for stage Ⅰ, four or five for stage Ⅱ, and six for stage ⅢA disease. Therefore, for the entire cohort, the number of examined LNs should be at least six, which is exactly consistent with the American Joint Committee on Cancer criteria.CONCLUSION The examination of higher numbers of LNs is associated with improved survival after resection surgery for N0 GBC. The guidelines for GBC surgery, which recommend that six LNs be examined at least, are statistically valid and should be applied in clinical practice widely.

Efficacy and safety of bevacizumab plus chemotherapy in Chinese patients with metastatic colorectal cancer:a randomized phase Ⅲ ARTIST trial

The efficacy and safety of bevacizumab with modified irinotecan,leucovorin bolus,and 5-fluorouracil intravenous infusion(mIFL) in the first-line treatment of metastatic colorectal cancer(mCRC) has not been well evaluated in randomized clinical trials in Chinese patients.We conducted a phrase Ⅲ trial in which patients with previously untreated mCRC were randomized 2:1 to the mIFL [irinotecan(125 mg/m2),leucovorin(20 mg/m2) bolus,and 5-fluorouracil intravenous infusion(500 mg/m2) weekly for four weeks every six weeks] plus bevacizumab(5 mg/kg every two weeks) group and the mIFL group,respectively.Co-primary objectives were progression-free survival(PFS) and 6-month PFS rate.In total,214 patients were enrolled.Our results showed that addition of bevacizumab to mIFL significantly improved median PFS(4.2 months in the mIFL group vs.8.3 months in the bevacizumab plus mIFL group,P < 0.001),6-month PFS rate(25.0% vs.62.6%,P < 0.001),median overall survival(13.4 months vs.18.7 months,P = 0.014),and response rate(17% vs.35%,P = 0.013).Grades 3 and 4 adverse events included diarrhea(21% in the mIFL group and 26% in the bevacizumab plus mIFL group) and neutropenia(19% in the mIFL group and 33% in the bevacizumab plus mIFL group).No wound-healing complications or congestive heart failure occurred.Our results suggested that bevacizumab plus mIFL is effective and well tolerated as first-line treatment for Chinese patients with mCRC.Clinical benefit and safety profiles were consistent with those observed in pivotal phase Ⅲ trials with mainly Caucasian patients.

Vitrectomy combined with silicone oil tamponade in the treatment of severely traumatized eyes with the visual acuity of no light perception

AIM: To evaluate the efficacy of surgical treatment of vitrectomy combined with silicone oil tamponade in the treatment of severely traumatized eyes with the visual acuity of no light perception (NLP).METHODS: This was a retrospective uncontrolled interventional case-series of 19 patients of severely traumatized eyes with NLP who underwent vitrectomy surgery at the Affiliated Hospital of Medical College, Qingdao University (Qingdao, China) during a 3-year period. We recorded perioperative factors with the potential to influence functional outcome including duration from the injury to intervention; causes for ocular trauma; open globe or closed globe injury; grade of vitreous hemorrhage; grade of endophthalmitis; grade of retinal detachment; size and location of intraocular foreign body (IOFB); extent and position of retinal defect; grade of proliferative vitreoretinopathy (PVR); type of surgery; perioperative complications and tamponade agent. The follow-up time was from 3 to 18 months, and the mean time was 12 months.RESULTS: After a mean follow-up period of 12 months (3-18 months) 10.53% (2/19) of eyes had visual acuity of between 20/60 and 20/400, 52.63% (10/19) had visual acuity less than 20/400 but more than NLP, and 36.84% (7/19) remained NLP. Visual acuity was improved from NLP to light perception (LP) or better in 63.16% (12/19) of eyes and the rate of complete retinal reattachment was 73.68% (14/19). Good visual acuity all resulted from those patients of blunt trauma with intact eyewall (closed globe injury). The perioperative factors of poor visual acuity prognosis included delayed intervention; open globe injury; endophthalmitis; severe retinal detachment; large IOFB; macular defect; a wide range of retinal defects andsevere PVR.CONCLUSION: The main reasons of NLP after ocular trauma are severe vitreous hemorrhage opacity; refractive media opacity; retinal detachment; retinal and uveal damages and defects, especially defects of the macula; PVR and endophthalmitis. NLP after ocular trauma in some cases does not mean permanent vision loss. Early intervention of vitrectomy combined with silicone oil tamponade and achieving retinal reattachment of the remaining retina, may make the severely traumatized eyes regain the VA of LP or better.

Expression of dectin-1 during fungus infection in human corneal epithelial cells

AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.

Effect of corneal graft diameter on therapeutic penetrating keratoplasty for fungal keratitis

AIM: To evaluate the effect of corneal graft diameter on therapeutic penetrating keratoplasty(PKP) for fungal keratitis. METHODS: A total of 116 patients (116 eyes) suffered from fungal keratitis underwent PKP at the Affiliated Hospital of Medical College Qingdao University from May 2006 to May 2010. They were divided into two groups according to the corneal graft diameter. 64 eyes' corneal graft diameter was 8.00mm or larger and 52 eyes' graft diameter was smaller than 8.00mm. The follow-up time was 2 years. The postoperative visual acuity and complications were documented and compared. RESULTS: Sixty-two (96.88%) eyes and fifty (96.15%) eyes preserved eyeballs respectively in two groups. There was no statistical difference in postoperative visual acuity (P = 0.961), corneal graft dear rate (P=0.132) or the incidence of recurred fungal infection (P=0.770) between two groups. But there was a higher incidence of graft rejection (P=0.020) and secondary glaucoma (P=0.039) in group with corneal graft diameter 8.00mm or larger. CONCLUSION: PKP is an effective treatment approach for fungal keratitis. There is a higher incidence of complications in large-diameter PKP for fungal keratitis.Effective, preventive and therapeutic measures can improve the prognosis.

Dectin-1 expression at early period of Aspergillus fumigatus infection in rat's corneal epithelium

AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat’s corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats’. There was no significantly difference of dectin-1 mRNA expression in group A and B (P 】0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P 【0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat’s corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P【0.01). · CONCLUSION:Dectin-1 exists in rat’s cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

Application of the revised Tumour Node Metastasis （TNM） staging system of clear cell renal cell carcinoma in eastern China： advantages and limitations

This study was designed to evaluate whether the revised 2010 Tumour Node Metastasis （TNM） staging system could lead to a more accurate prediction of the prognosis of renal cell carcinoma （RCC） patients. A total of 1216 patients who had undergone radical nephrectomy or partial nephrectomy for RCC from 2003 to 2011 were enrolled. All of the patients had pathologically confirmed clear cell RCC （ccRCC）. All cases were staged by both the 2002 and 2010 TNM staging systems after pathological review, and survival data were collected. Univariate and multivariate Cox regression models were used to evaluate cancer-specific survival （CSS） and progression-free survival （PFS） after surgery. Continuous variables, such as age and tumour diameter, were calculated as mean values and standard deviations （s.d.） or as median values. Survival was calculated by the Kaplan-Meier method, and the log-rank test assessed differences between groups. Statistically significant differences in CSS and PFS were noted among patients in T3 subgroups using the new 2010 staging system. Therefore, the revised 2010 TNM staging system can lead to a more accurate prediction of the prognosis of ccRCC patients. However, when using the revised 2010 staging system, we found that more than 92% of patients （288/313） with T3 tumours were staged in the T3a subgroup, and their survival data were not significantly different from those of patients with T2b tumours. In addition, T2 subclassification failed to independently predict survival in RCC patients.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B (PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CEC, which promotes the viability and proliferation of cells.

Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND： Hypoxia inducible factor-1 alpha （HIF-1 （x） and erythropoietin（EPO）, possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance （IT）. OBJECTIVE：To observe the neuroprotective effect of cerebral ischemic preconditioning（IPC） of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN ： A randomized and controlled observation SETTING： Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University MATERIALS： Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd （Wuhan）; Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company （USA）. METHODS： This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot： IPC group （n=40）, sham-operation group （n=40） and control group （n=4）. In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1^st, 3^rd, 7^th, 14^th and 21^st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion （MCAO）. That was to say, after 10-minute preischemia, suture was exited to the extemal carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation（only common carotid artery and crotch were exposed, and MCAO by suture was omitted）, and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume （The total cerebral infarction area of each layerxinterspace ） was calculated. After brain tissue was stained by haematoxylin-esoin （HE）, the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α（and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES： ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS：Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group： Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively [（161.2±6.9） mm^3 vs （219.9±11.2） mm^3, （134.9±9.0） mm^3 vs （218.6±13.0） mm^3, （142.9±13.7） mm^3 vs （221.3±14.2） mm^3, t=-8.924, 10.587,7.947, P〈 0.01]. ② Observation of nerve cell form of brain tissue： HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus ： The expression of HIF-1αof IPC group was significantly higher than that of sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively （125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763,9.899,P 〈 0.05-0.01）. The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3^rd and 7^th days after perfusion respectively （141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18,t=5.499,3.970, P〈 0.05）. CONCLUSION ： ①IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.

Outcomes and prognostic factors of patients with stage? ⅠB and? ⅡA pancreatic cancer according to the 8^(th) edition American Joint Committee on Cancer criteria

AIM To evaluate the changes in the 8^(th) edition American Joint Committee on Cancer(AJCC) for defining stage?ⅠB and?ⅡA pancreatic cancer and identify their prognostic factors.METHODS Pancreatic cancer patients were selected from the Surveillance Epidemiology and End Results database(1973-2013). The enrolled patients were divided into?ⅠB and?ⅡA groups based on tumor size according to the 8^(th) edition AJCC criteria. Clinical characteristics, including age, gender, race, tumor size, primary site, and grade were summarized. Univariate and multivariate analyses were performed to explore the prognostic factors of the?ⅠB and?ⅡA stages of pancreatic cancer under new criteria.RESULTS A total of 1349 pancreatic cancer patients were included. More patients had stage?ⅠB rather than stage?ⅡA. Stage?ⅠB tumors(54.85%) were mainly located in the head of the pancreas, while stage?ⅡA tumors were more often located in the tail and head of the pancreas(35.21% and 31.75%, respectively). The survival time of stage?ⅠB and?ⅡA patients had no significant difference. Univariate and multivariate analyses indicated that the prognostic factors of survival for stage?ⅠB and?ⅡA patients were different. for stage?ⅠB patients, age and primary site were the independent prognostic factors; for stage?ⅡA patients, age and grade were the independent prognostic factors. The risk of death was lower among patients aged ≤ 65 years than those aged > 65 years.CONCLUSION The prognostic factors for stage?ⅠB and?ⅡA patients are different, but age is the independent prognostic factor for all patients. The survival time of stage?ⅠB and?ⅡA patients has no significant difference.

Malignant meningioma of the cerebellopontine angle in a 2-year-old girl:a case report and literature review

Meningioma is a common intracranial tumor in adults.Pediatric cases account for approximately 1.5% of all intracranial meningiomas,and very few cases show malignant histological features.Primary pediatric malignant meningioma in the cerebellopontine angle is extremely uncommon.Herein,we report a 2-yearold girl with malignant meningioma in the cerebellopontine angle.The clinical features,diagnosis,and treatment protocol are discussed.

Primary Endodermal Sinus Tumor in the Posterior Cranial Fossa:Clinical Analysis of 7 Cases

Objective To clarify the clinical features,therapeutic method and outcomes of the primary endodermal sinus tumors(ESTs)in the posterior cranial fossa.Methods The English literatures on EST in the posterior cranial fossa were retrieved from PubMed and reviewed.And a 4-year-old boy diagnosed with EST in our hospital was reported.The clinical manifestations,therapy,pathologic features,and prognosis of these cases were analyzed.Results Only seven cases of the ESTs in the posterior cranial fossa were enrolled in this review,including six cases searched from the PubMed and one case from our hospital.Six patients were boy and one patient’s gender was not available from the report.Ages ranged from 1 to 5 years(mean 3.14 years).The mean tumor size in our cohort was 4.4 cm.Six cases came from East Asia.Schiller-Duval bodies were found in all seven neoplasms.All tumors were positive for alpha-fetoprotein.The alpha-fetoprotein level in serum was increased to a very high level before therapy and depressed quickly after the effective chemotherapy.The mean follow-up time was 24.4 months(range 5-52 months).Six tumors were totally removed,and four of them recurred.Three cases died including one whose tumor was partially removed.Conclusions The serum alpha-fetoprotein level is well correlated with the severity of the tumor.A combination of operation and chemotherapy might be the effective management for EST in the posterior cranial fossa.The prognosis of extragonadal intracranial EST is poor.

Inflammatory Myofibroblastic Tumor Mimicking Malignant Meningioma in the Middle Cranial Fossa:a Case Report

INFLAMMATORY myofibroblastic tumor (IMT),also known as inflammatory pseudotumor or plasma cell granulomas,is an uncommon fibro-inflammatory lesion which is composed of inflammatory cells and myofibroblastic spindle cells.

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be Expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. ' METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic Expression vector was transferred into cat corneal endothelial cells by Effectene (TM) lipofectine. The transfection efficiency of Effectene (TM) lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MU) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene (TM) lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly Expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Polymorphisms of ERCC1, XPD, XRCC1 and XPG Predict Clinical Outcome in Advanced Gastric Cancer Patients Receiving Oxaliplatin-Based Chemotherapy in Chinese Population

OBJECTIVE To investigate whether polymorphisms in ERCC1,XPD,XPG,XRCC1 genes are associated with clinical outcomes inadvanced gastric cancer (AGC) patients treated with oxaliplatin-based chemotherapy.METHODS The genetic polymorphisms in ERCC1,XPD,XPG,XRCC1 were determined in 94 advanced gastric cancer patientstreated with oxaliplatin-based chemotherapy,using TaqMan-MGBprobes.The clinical response of 60 patients with stage Ⅳ disease,time to progression (TTP) and overall survival (OS) of 94 patientswere evaluated.RESULTS The overall disease control rate (CR + PR + SD) of the60 patients in stage Ⅳ was 70% (42/60).Patients with XRCC1 399G/G,XPG 46 C/C genotypes showed enhanced response to theoxaliplatin-based chemotherapy compared to those with othergenotypes (P<0.05).The median OS and TTP of the patientswere 5.5 months and 9.0 months,respectively.Among the 4types of polymorphisms in the study,XRCC1 399 G/A + A/A,XPG 46 C/T + T/T genotypes were regarded to be associatedwith chemoresistance and poor survival (P<0.05).Combinationanalysis of the 2 polymorphisms using the Kaplan-Meier methodrevealed that the TTP and OS of the patients with a number of riskgenotypes were significantly shortened (P<0.05).No significantassociation was found between the genotypes of the XPD codon751,the ERCC1 codon 118 and the clinical outcome (P>0.05).CONCLUSION Testing for XRCC1 399,XPG 46 polymorphismsmay allow identification of the gastric cancer patients whowill benefit from oxaliplatin-based chemotherapy.Specificpolymorphisms may influence clinical outcomes of AGCpatients.Selecting specific chemotherapy based on pretreatmentgenotyping represents an innovative strategy that warrantsprospective studies.

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

Profiles of metabolic gene expression in the white adipose tissue, liver and hypothalamus in leptin knockout(Lep^(△I14/△I14)) rats

Leptin deficiency is principally linked to metabolic disorders. Leptin knockout（Lep△I14/△I14 Sprague Dawley rats created by CRISPR/Cas9 is a new model to study metabolic disorders. We used a whole rat genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of the white adipose tissue, liver and hypothalamus in Lep△I14/△I14）and wild-type（WT） rats. We found 1,651 differentially expressed（enriched） genes in white adipose tissue,916 in the liver, and 306 in the hypothalamus in the Lep△I14/△I14 rats compared to WT. Gene ontology category and KEGG pathway analysis of the relationships among differentially expressed genes showed that these genes were represented in a variety of functional categories, including fatty acid metabolism, molecular transducers and cellular processes. The reliability of the data obtained from microarray was verified by quantitative real-time PCR on 14 representative genes. These data will contribute to a greater understanding of different metabolic disorders, such as obesity and diabetes.