Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Effects of Roudoukou-8 San Extract on Hydrogen Peroxide Induced Cardiomyocyte Injury

[Objectives] To study the effects of Roudoukou-8 San extract on hydrogen peroxide( H_2O_2) induced cardiomyocyte injury of rats and explore its action mechanism. [Methods] The cardiomyocytes of neonatal rats were isolated and cultured,and the H_2O_2 induced cardiomyocyte injury model was established. Methyl thiazolyl tetrazolium( MTT) assay was used to detect the protective effects of Roudoukou-8 San extract on H_2O_2 induced cardiomyocyte. The effects of Roudoukou-8 San on cardiomyocyte morphology were observed under inverted microscope. The contents of lactate dehydrogenase( LDH),creatine kinase( CK) and aspartate aminotransferase( AST) in cell culture medium were determined by automatic biochemical instrument; the levels of malondialdehyde( MDA),superoxide dismutase( SOD) and nitric oxide( NO) in the cells were detected by kit method. The apoptotic morphology of cardiomyocytes was observed by Hoechst fluorescence staining.Cell apoptosis were measured by Annexin V and PI double staining and flow cytometer. [Results]100 μmol/L of H_2O_2 acting 2 h could cause about 50% of cardiomyocyte injury. H_2O_2 model group showed increased cell gap,decreased cell count,cell cytoplasmic vacuoles and other obvious damages. Compared with H_2O_2 model group,in Roudoukou-8 San extract dose groups,cardiomyocyte morphology showed different degrees of improvement,Roudoukou-8 San extract can significantly reduce the content of LDH,CK and AST in H_2O_2 injured myocardial cell culture solution,significantly reduce content of MDA and NO in H_2O_2 injured myocardial cells,increase the SOD activity,and significantly inhibit the apoptosis of H_2O_2 injured myocardial cells. [Conclusions]Through improving the cell survival status,increasing the cell viability,reducing oxidative stress injury,inhibiting inflammatory responses,and inhibiting apoptosis,Roudoukou-8 San extract can improve the state of H_2O_2 induced cardiomyocyte injury,so as to protect H_2O_2 injured myocardial cells.

Application of malignant pleural effusion cell blocks in the diagnosis and personalized treatment of advanced non-small cell lung carcinoma

Objective The aim of the study was to investigate the efficacy of immunocytochemistry and related gene detection using cell block for the diagnosis and individualized treatment of advanced lung cancer.Methods Sixty-five malignant pleural effusion specimens were collected to make cell blocks, which were used for hematoxylin and eosin(H&E) staining, immunocytochemical studies, and gene sequencing of the tumors to guide the individualized diagnoses and treatment of the given tumors. Results The tumor cells in the cell block sections were abundant in number with high quality cellular structures, and the histological morphological characteristics were partially maintained. Immunocytochemical staining was helpful in identifying the cell origin and tumor classification, and amplification refractory mutation system(ARMS) was used to determine the mutation status of epidermal growth factor receptor(EGFR). Of the 65 samples, 50 had a diagnosis of adenocarcinoma, 7 were pulmonary squamous cells, 6 were small cell carcinoma of the lung, and 2 were mesothelioma. The morphological features of the tumors were as follows: acinar formation, papillary and single cells for adenocarcinoma;intercellular bridges for squamous cell carcinoma;and morphology of the small cells is similar to that of the smear. Correlating with the results of immunocytochemical staining and clinical data analysis, 40 cases were confirmed as pulmonary adenocarcinoma, with an additional 4 cases of breast cancer, 3 cases of ovarian adenocarcinoma, and 3 cases of colorectal adenocarcinoma. Of the 47 non-small cell lung carcinoma(NSCLC) patients, EGFR mutations were detected in 26 cases(55.3%) by ARMS, with four mutation types: exon 19 deletion(13 cases, 50.0%), exon 2l point mutations L858R(11 cases, 42.3%) and L861Q(1 case, 3.8%), and exon 18 point mutation G719X(1 case, 3.8%). Conclusion Malignant pleural effusion cell blocks combined with immunocytochemical markers and molecular pathology are helpful for the diagnosis of advanced tumors, the identification of tumor properties and histological tumor origin, and the selection of individualized treatment for advanced lung cancer.

Genetic association of urokinase-type plasminogen activator gene rs2227564 site polymorphism with sporadic Alzheimer's disease in the Han Chinese population

A missense C/T polymorphism in exon 6 （the NCBI rslD is rs2227564） of the urokinase-type plasminogen activator gene has been identified as a possible hot spot for Alzheimer＇s disease risk. The present study analyzed urokinase-type plasminogen gene polymorphisms of rs2227564 with sporadic Alzheimer＇s disease by PCR-restriction fragment length polymorphism. Results showed that CC, CT and TT genotype distribution frequencies had significant differences between sporadic Aizheimer＇s disease patients and healthy controls, in-depth analysis of the association between urokinase-type plasminogen gene rs2227564 polymorphisms and sporadic Alzheimer＇s disease indicated that people with the C-positive genotype CC ＋ CT were at a higher risk for developing sporadic Alzheimer＇s disease. These results support the contribution of the polymorphisms of rs2227564 in the urokinase-type plasminogen gene to the pathogenesis of sporadicAIzheimer＇s disease in the Han Chinese population.

Safety and efficacy of multi-kinase inhibitor plus endostar treatment in patients with metastatic renal cell carcinoma and pleural effusion

Objective The aim of this study was to evaluate the safety and efficacy of multi-kinase inhibitor plus endostar treatment in patients with metastatic renal cell carcinoma(m RCC) and pleural effusion. Methods A total of 10 patients with m RCC(8 clear-cell RCCs, 1 papillary RCC, 1 chromophobe RCC) with pleural effusion from January 2014 to October 2015 were recruited. Four patients received sorafenib(400 mg, twice daily), while six received sunitinib(50 mg, once daily; 2 weeks on and 1 week off). All patients received multi-kinase inhibitor plus pleural cavity perfusion of endostar(15 mg on days 1–4 for 1 or 2 weeks). Results The response rate of pleural effusion was 70%. Adverse reactions were limited and mild.Conclusion The regimen of multi-kinase inhibitor plus pleural cavity perfusion of endostar was both effective and safe for the treatment of patients with m RCC with pleural effusion, and may control local symptoms.

miR-31 inhibits proliferation and promotes apoptosis of osteosarcoma cells through PAX9

Objective: To investigate the role of microRNA-31 (miR-31) in osteosarcoma and the molecular mechanism of miR-31 in proliferation and apoptosis of osteosarcoma. Methods:real-time quantitative PCR (qRT-PCR) was used to detect the expression of miR-31 in human osteosarcoma. Pearson 's chisquared was applied to detect the correlation between miR-31 and clinicopathological features. CCK-8 and ANNEXIN-FITC/PI double staining were used to detect the proliferation and apoptosis of osteosarcoma cells. Results: The results of qRT-PCR showed that the expression of miR-31 was down-regulated in osteosarcoma tissues compared with matched precancerous bone tissues. The level of miR-31 in osteosarcoma tissues with high-grade malignancy and poor overall survival was even lower. CCK-8 and Annexin-FITC/PI double staining assays showed that miR-31 inhibited proliferation and promoted apoptosis of osteosarcoma cells. According to luciferase assay, miR-31 inhibited PAX9 expression directly. And PAX9 promoted proliferation and inhibited apoptosis of osteosarcoma cells. Conclusion:miR-31 inhibits proliferation and promotes apoptosis of osteosarcoma cells by targeting PAX9. miR-31 and PAX9 can be used as therapeutic targets for osteosarcoma.

Expression and effect of proline hydroxylase domain 2 in retina of diabetic rats

AIM： To observe the expression of proline hydroxylase domain 2 （PHD2） in the retina of diabetic rats and investigate the relationship between PHD2 and relevant intraocular vascular proliferation factors, METHODS： Sixty male specific pathogen free （SPF） Sprague-Dawley （SD） rats were randomly divided into two groups： the diabetic group and the control group. The rats in the diabetic group were intraperitoneally injected with 60 mg/kg （0.60 mL/100 g） of streptozotocin to induce a diabetic rat model. The rats in the control group were injected with an equal volume of sodium citrate buffer solution by the same method. Hematoxylin- eosin （HE） staining and immumofiuorescence （IF） method were adopted to observe the pathological changes of retinal tissues and the expression of PHD2, glial fibrillary acidic protein （GFAP）, vascular endothelial growth factor （VEGF） by 8wk. RT-PCR method was applied to detect the expressions of mRNA of PHD2, VEGF and GFAP. The relationship between PHD2 and other vascular proliferation factors was analyzed. RESULTS： HE staining showed that there was the retinal tissue edema in the diabetic group, and the arrangement was in disorder, and proliferation could be seen. IF staining： in the retina of normal rats, PHD2 was not expressed, GFAP and VEGF were mainly expressed in astrocytes; while in the diabetic rats, PHD2, GFAP and VEGF staining showed strong positivity in all retinal layers, mainly in neurogliocytes. PHD2 was co-expressed with VEGF and GFAP. The mRNA expression levels of PHD2, GFAP and VEGF in the diabetic group were obviously higher than that in the control group, respectively 1.83 times, 1.75 times and 2.08 times. The difference had statistical significance （P〈0.01）. CONCLUSION： The high expression of PHD2 in the retina of early-stage diabetic rats might result from secretion of neurogliocytes induced by local high- concentration blood glucose, thus promoting the expression of VEGF and GFAP, PHD2 plays an important role during the occurrence of diabetic retinopathy.

Relationship between molecular changes in epidermal growth factor receptor(EGFR)and anaplastic lymphoma kinase(ALK)mutations in lung adenocarcinoma

Objective This study aimed to analyze the relationship between the mutations in epidermal growth factor receptor(EGFR)and anaplastic lymphoma kinase(ALK)and their impact on the prognosis and treatment of lung adenocarcinoma.Methods A total of 158 cases of lung adenocarcinoma reported between January 2007 and January 2014 were retrospectively analyzed.These tumors were resected using radical pneumonectomy and underwent pathology-based diagnosis at our institution(Inner Mongolia People’s Hospital,Hohhot,China).The tissue sections were evaluated using the updated World Health Organization classification of lung adenocarcinomas(2015 version),with each histological component recorded in 5%increments.The histological subtypes were classified,and any surviving cases were followed up.The reverse transcription-polymerase chain reaction(RT-PCR)and direct DNA sequencing were used to evaluate mutations in exons 18,19,20,and 21 in the EGFR gene,and the echinoderm microtubule-associated protein-like 4 gene-ALK variant(EML4-ALK)fusions were detected using sequencing.Results Our cohort included 25 patients with pre-invasive adenocarcinoma,13 patients with lepidic,66 patients with acinar,13 patients with papillary,and 25 patients with solid infiltrative adenocarcinoma with the remaining cases presenting with a variety of pathological subtypes.The prognosis of each histological subtype was different with the 5-year disease-free survival and 5-year overall survival(OS)of pre-invasion adenocarcinoma at 100%;the 5-year OS of lepidic,acinar,and papillary adenocarcinoma patients was only 84.6%,72.7%,and 76.9%,respectively.The 5-year OS of solid and mucinous adenocarcinomas were 32.0%and 36.4%,respectively.EGFR mutation was detected in 69 cases with a mutation rate of 43.7%and majority of these mutations were found in exons 19(50.6%)and 21(37.9%),with women and non-smokers shown to experience a higher mutation rate(P<0.05).However,histological subtype analysis showed that EGFR mutations were primarily found in adenocarcinomas.Most of these mutations were found in lepidic(53.8%)or acinar adenocarcinomas(50.0%),whereas these mutations were rare in both solid(28.0%)and mucinous adenocarcinoma(27.2%).The fusion mutation rate in the EML4-ALK gene was 5.69%,and was most common in young,nonsmoking patients(P<0.05).Conclusion The prognosis of patients in each lung adenocarcinoma subtype is different,and these outcomes are likely related to mutations in the EGFR and EML4-ALK genes.EGFR mutation rates are higher in lepidic and acinar adenocarcinomas,whereas EML4-ALK gene fusion mutations are more common in solid and mucinous adenocarcinoma.EGFR mutations are more common in female and non-smoking patients,whereas EML4-ALK fusions are more common in young,non-smoking patients.

The efficacy of capecitabine and temozolomide against neuroendocrine carcinomas

Objective Neuroendocrine carcinomas(NECs) are resistant to currently available chemotherapy agents, and its therapeutic options are limited. Preclinical data have suggested synergy between capecitabine and temozolomide(CAPTEM). Therefore, we evaluated the efficacy and safety of CAPTEM in patients with metastatic NECs who have failed prior therapies.Methods A retrospective review was conducted on seven patients with metastatic NECs for whom platinum-based chemotherapies and hepatic chemoembolization failed. Patients received capecitabine(1000 mg twice daily on days 1-14) and temozolomide(150–200 mg/m^2 once daily on days 10–14) every 28 days. Tumor assessments were performed every two cycles.Results Among the seven patients treated, two achieved partial remission and four achieved stable disease. The total response rate was 29%, and the clinical benefit was 86%. Median progression-free survival was 10(range: 8–14) months. The most common toxicities were grade 1 and 2 neutropenia, grade 1 fatigue, and grade 1 and 2 hand-foot syndrome. No grade 4 toxicities or treatment-related deaths were observed.Conclusion Our study showed that the CAPTEM regimen is an effective and well-tolerated salvage option for NECs. Further prospective studies are warranted to evaluate optimal combinations of the CAPTEM regimen for NECs.

A Prospective Randomized Multicenter Controlled Trial on Salvianolate for Treatment of Unstable Angina Pectoris in A Chinese Elderly Population

Objective: To evaluate the efficacy and safety of salvianolate in elderly patients with unstable angina pectoris(UAP). Methods: A prospective double-blind randomized placebo-controlled multicenter trial in elderly patients with UAP from 13 third-grade class-A hospitals in China was performed. A total of 318 patients were randomly allocated in a 1:1 ratio to an experimental group(160 patients) and a control group(158 patients). The experimental group was treated with salvianolate for 14 days on the basis of conventional medicine, and the control group was given a placebo for 14 days with the same criteria. Follow-up was lasted 28 days in both groups. The primary endpoint was biweekly frequency of angina pectoris attacks. The secondary endpoints included biweekly dosage of nitroglycerin, the Seattle Angina Questionnaire, angina pectoris severity and duration, myocardial injury markers, high-sensitivity C-reactive protein(hs-CRP) and N-terminal pro-B-type natriuretic peptide(NT-proBNP), as well as major adverse cardiovascular events(MACEs). Safety was assessed according to adverse events and serious adverse events. Results: Baseline characteristics were similar between treatment groups. Compared with those in the control group, the frequency of biweekly angi na attacks(2.92 vs. 4.08, P =0.025), th e biweekly dosage of nitroglycerin, as well as the severity and duration of angina attacks(P <0.01) were reduced by salvianolate. The Seattle Angina Questionnaire score was also significantly improved in the experimental group than in the control group(P <0.05). No significant differences were observed between the two groups with respect to the incidence of MACEs. Salvianolate was well tolerated. Conclusions: Salvianolate appear to have efficacy and well tolerated for elderly patients with UAP.

经动脉介入化疗栓塞前后中晚期宫颈癌病变组织中Caspase-3的变化及临床意义

目的探讨中晚期宫颈癌子宫动脉介入化疗栓塞的近期临床疗效及其病变组织中Caspase-3表达的意义。方法收集2013年1月-2015年6月在我院行PEF化疗方案,采用双侧子宫动脉选择性化疗栓塞术治疗中晚期宫颈癌患者27例,根据患者的疗效和反应给予1-2个疗程介入化疗栓塞,具有手术指征者于末次介入化疗栓塞术后3周左右行广泛子宫切除＋盆腔淋巴结清扫手术。留取介入化疗栓塞前后宫颈癌组织蜡块标本,采用S-P免疫组织化学方法检测宫颈癌病变组织中Caspase-3的表达情况。结果介入化疗栓塞前后宫颈癌组织中Caspase-3的阳性表达率有差异。结论动脉介入化疗栓塞术可以缩小中晚期宫颈癌病灶体积,降低临床分期,增加中晚期宫颈癌患者手术机会,且动脉介入化疗栓塞后宫颈癌组织中Caspase-3蛋白表达上升,说明介入化疗栓塞通过促进宫颈癌细胞凋亡从而抑制癌细胞的生长;Caspase-3能在一定程度上预测肿瘤组织对介入化疗栓塞的敏感性及疗效。