Study on neoplasia of hydatidiform mole by detecting telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells

By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation between the expression level of hTERT mRNA and the prognosis of hydatidiform mole, and to evaluate the clinic value of quantitative determination of hTERT mRNA in the diagnosis of hydatidiform mole. Methods： A real-time fluorescent quantitative RT-PCR （FQ RT-PCR） assay based on TaqMan fluorescence methodology and the Light-Cycler system was used to quantify the full range of hTERT mRNA copy numbers in 30 samples of hydatidiform mole and the neoplasia of hydatidiform mole. The normalized hTERT （NhTERT） was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100x （hTERT/GAPDH） ratio. Based on the prognosis of the hydatidiform mole, the patients were divided into two groups： the experimental group and the control group, to compare the telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells. Results： hTERT mRNA was both expressed in the peripheral blood mononuclear cells and pathological tissues in the mole experimental group and the control group. In the mole experimental group, the values were 6.31±0.32 and 6.24±0.44, respectively, and there was no significant difference between them （P〉0.05）. But in the control group the values were 1.21±0.65 and 1.40±0.61, respectively, and there was no significant difference between them （P〉0.05） The values in experimental group was significantly higher than those in the control group （P〈0.01）. Conclusion： Quantitative determination of hTERT mRNA by FQ RT-PCR is a rapid and sensitive method, hTERT in peripheral blood mononuclear cells may have potential use as a biomarker for the early detection of the prognosis of the hydatidiform mole.

Msi1基因沉默对人肝癌HepG2细胞的影响

目的:探讨siRNA沉默Msi1基因表达后,人肝癌HepG2细胞的增殖与凋亡的情况。方法:设计合成针对Msi1 mRNA序列的siRNA多条序列,分别转染人肝癌HepG2细胞。使用RT–PCR方法检测各组细胞Msi1基因表达情况;Western blot法检测survivin蛋白、caspase3蛋白表达情况;MTT法、平皿克隆实验检测HepG2细胞增殖情况;流式细胞术检测各组HepG2细胞凋亡情况。结果:(1)RT-PCR结果:转染后24 h后,序列a,b,c转染效率分别为65%,64%及62%。其抑制率分别是Msi1 siRNAa(68%)、Msi1 siRNAb(56%)、Msi1 siRNAc(35%)。(2)流式细胞术检测各组细胞凋亡:空白组、阴性组、脂质体组、Msi1 siRNAa组凋亡率分别为(4.14±0.26)%、(4.51±0.78)%、(4.19±1.21)%,(16.8±0.26)%,Msi1 siRNAa组与空白对照组比较差异有统计学意义(P<0.05)。(3)Msi1 siRNAa最能有效抑制肝癌HepG2细胞中Msi1表达,与空白对照组比较,Msi1 siRNAa组HepG2细胞生长、增殖速度明显减缓(P<0.05);survivin蛋白表达水平显著下调(P<0.05),而caspase3蛋白表达水平上调(P<0.05);Msi1 siRNAa组凋亡率增高(P<0.05)。结论:沉默Msi1可抑制人肝癌HepG2细胞的增殖,诱导其凋亡。

Isolation of the drought up-regulated gene in Alternanthera philoxeroides by differential display technique

Under drought stress, adventitious roots of Alternanthera philoxeroides seedlings will grow long thick fleshy roots, which are assumed to improve performance of the plant by more efficient reservation and absorption of water from deep soil layers. In this study, the differential display technique was used to clone morphogenesis-related genes from A. philoxeroides roots treated with drought, which would help to improve crop plants＇ drought-tolerance by transgenic method; by 15 pairs of primer combinations, twenty putative drought up-regulated gene segments induced by drought were obtained; and one of them was confirmed by reverse northern blot, and subsequently cloned and sequenced. A homologous analysis revealed that it might be a new sequence. Semi-quantitative RT-PCR analysis showed that the gene was up-regulated by drought and salt stress.

Isolation and Expression Analysis of MaPRMT1 Gene in Banana

[Objective] The aim of experiment was to lay molecular foundation for studying maturity mechanism of banana after harvest. [Method] The combined method of suppressing subtractive hybridization and cDNA micro-array were used to obtain cDNA segment of one PRMT gene in banana and the whole cDNA sequence of the gene was cloned.The bioinformatics analysis was operated on it,in addition, the expression profile analysis was conducted in different organs and different mature periods of banana.[Result] The whole length of cDNA in MaPRMT1 was 1 158 bp and possessed a complete open reading frame,which could encode 385 amino acids.It had high homology with PRMT in plant,containing one Methyltransf_1 domain.The MaPRMT1 gene was expressed in root,stem,leaf and fruit of banana and the expression levels in stem and leaf were relatively high.As the increase of days after harvest,the expression level declined gradually,however it reached maximum when ethylene release was biggest,then it declined.[Conclusion] MaPRMT1 belonged to the first kind of arginine methyltransferase and it was expressed differently in different organs and fruits at different mature periods.

JNK3 involvement in nerve cell apoptosis and neurofunctional recovery after traumatic brain injury

Increasing evidence has revealed that the activation of the JNK pathway participates In apoptosis o1 nerve cells and neurological function recovery after traumatic brain injury. However, which genes inI the JNK family are activated and their role in traumatic brain injury remain unclear. Therefore, in this study, in situ end labeling, reverse transcription-PCR and neurological function assessment were adopted to investigate the alteration of JNK1, JNK2 and JNK3 gene expression in cerebral injured rats, and their role in celt apoptosis and neurological function restoration. Results showed that JNK3 expression significantly decreased at 1 and 6 hours and 1 and 7 days post injury, but that JNK1 and JNK2 expression remained unchanged. In addition, the number of apoptotic nerve cells surrounding the injured cerebral cortex gradually reduced over time post injury. The Neurological Severity Scores gradually decreased over 1,3, 5, 14 and 28 days post injury. These findings suggested that JNK3 expression was downregulated at early stages of brain injury, which may be associated with apoptosis of nerve cells. Downregulation of JNK3 expression may promote the recovery of neurological function following traumatic brain injury.

Detection of Grass Carp Hemorrhage Virus (GCHV) from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as "Red Spot Disease", is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and comparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplification. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain induces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the 6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines, including CAB, FHM, CIK, EPC, CCO and GCO, infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHV strains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and the two strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-V viruses have been replicated and amplified despite there being no cytopathogenic effects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published recently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp fragment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is identical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously, the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product, and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.

Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats

Acupuncture for the treatment of Parkinson＇s disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu （GV16） and Taichong （LR3） acupoints in rat models of Parkinson＇s disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson＇s disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson＇s disease. Thus, electroacupuncture may be useful in the treatment of Parkinson＇s disease.

Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR （FQ RT-PCR）. The results showed that the activities of creatine kinase （CK） and glutamic-pyruvic transaminase （GPT） were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 （HSC70） mRNA was changeable. The transcription of heat shock protein 70 （HSPT0） mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

Increased CD133^+ cell infiltration in the rat brain following fluid percussion injury

The prominin-1/CD133 epitope is expressed in craniocerebral trauma in animal models of fluid undifferentiated cells. Studies have reported that percussion injury induces production of a specific stem cell subgroup. It has been hypothesized that fluid percussion injury induces CD133＋ cell infiltration in the brain tissue. The present study established a traumatic brain injury model through fluid percussion injury. Immunohistochemical staining showed significantly increased CD133 antigen expression in the rat brain following injury. CD133＋ cells were mainly distributed in hippocampal CA1 3 regions, as well as the dentate gyrus and hilus, of the lesioned hemisphere. Occasional cells were also detected in the cortex. In addition, reverse transcription-PCR revealed that no change in CD133 mRNA expression in injured brain tissue. These results suggested that fluid percussion injury induced CD133 antigen expression in the brain tissues as a result of conformational epitope changes, but not transcriptional expression.

Effect of sericin on diabetic hippocampal growth hormone/insulin-like growth factor 1 axis

Previous studies have shown that sericin extracted from silk cocoon significantly reduces blood glucose levels and protects the nervous system against diabetes mellitus. In this study, a rat type 2 diabetes mellitus model was established by intraperitoneal injection of 25 mg/kg streptozotocin for 3 successive days, following which the rats were treated with sericin for 35 days. After treatment, the blood glucose levels of the diabetic rats decreased significantly, the growth hormone level in serum and its expression in the hippocampus decreased significantly, while the insulin-like growth factor-1 level in serum and insulin-like growth factor-1 and growth hormone receptor expression in the hippocampus increased significantly. The experimental findings indicate that sericin improves disorders of the growth hormone/insulin-like growth factor 1 axis to alleviate hippocampal damage in diabetic rats.

Polypeptide Daintain as a New Biomarker for Detecting Breast Tumor

Daintain, a novel bioactive peptide produced and secreted by macrophages, was expressed in breast tumor tissues. The spatial distributions of daintain in 66 breast tumor specimens were investigated with immuno-histochemistry method. Reverse transcription polymerase chain reaction （RT-PCR） and in situ hybridization inspection system were also used to detect daintain in 45 cases of malignant breast tumors. The final results show that 93% high positive responses to daintain on breast cancer tumors. RT-PCR demonstrated that, no smear of daintain transcripted in benign tissues was found, and light smear in peri-cancer tissue was observed. Distribution of daintain was distinguishable among benign tissues, hyperplasia tissues, immature hyperplasia and invasive breast cancer, which can be used to mark the progression of the malignant lesion development. We conclude that the expression of daintain is up-regulated in breast cancers, which indicates that the peptide is closely associated with the disease progression. So daintain could be used as the biomarker for detecting breast cancer.

The detection of micrometastases in the peripheral blood of patients with breast cancer for hSBEM mRNA and CD44V6 mRNA

Objective： Successful treatment of breast cancer greatly depends on the early detection of its metastasis, therefore a sensitive and specific biomarker for detecting dissemination of the cancer cells will help to achieve this goal. This study was to evaluate the prognostic significance of human small breast epithelial mucin （hSBEM） and CD44V6 in breast cancer. Methods： The expressions of hSBEM mRNA and CD44V6 mRNA were detected with nested reverse transcription polymerase chain reaction （nested RT-PCR） in 67 samples of breast cancer and adjacent normal breast tissue, 16 samples of breast benign lesions tissue, and 67 specimens of peripheral blood from patients with breast cancer, 16 specimens of benign breast lesions, 20 specimens of healthy volunteers, and 25 （each five cases） other carcinomas tissue samples, including those of gastric carcinoma, colorectal carcinoma, esophageal carcinoma, lung carcinoma, and ovary carcinoma, were analyzed for hSBEM mRNA expression by nested RT-PCR. Results： hSBEM mRNA expression was observed in 62/67 （92.54%） of breast cancer, 14/16 （87.50%） of breast benign lesions and 59/67 （88.05%） of normal breast tissue, with no significant differences between them （P 〉 0.05）. None of the samples from other cancer tissues were positive. In peripheral blood the expression of hSBEM mRNA was detected in 34/67 （50.75%） from patients with breast cancer, with significant increasing （P 〈 0.05） in the cases of metastatic disease （stage Ⅳ） and those with lymph node metastasis compared with localized disease （stage Ⅰ, Ⅱ and Ⅲ） and without lymph node metastasis, but its expression was not found in peripheral blood of patients with benign breast lesions or healthy volunteers. Although CD44V6 mRNA was significantly higher in breast cancer than in benign breast lesions tissue and normal breast tissue, its expression in peripheral blood show no significant difference （P 〉 0.05） in the patients with breast cancer （82.09%）, benign breast lesion （75.00%）, or healthy volunteers （70.00%）. The expressions of hSBEM mRNA and CD44V6 mRNA had no correlation with the age of the patients, size of primary tumor, histological type and estrogen or progestin receptor status （P 〉 0.05）. Conclusion： hSBEM mRNA, as assessed by nested RT-PCR, shows a mammary-specific and mammary-sensitive expression, and is a sensitive indicator of hematogeneous spread of breast cancer cell, while CD44V6 shows low sensitivity and specificity in detecting dissemination of breast cancer cell in peripheral blood, hSBEM mRNA is a promising molecular biomarker for detecting breast cancer micrometastases.

Abnormal expression of VEGF and its gene transcription status as diagnostic indicators in patients with non-small cell lung cancer

Objective Angiogenesis is known to be essential for the survival,growth,invasion,and metastasis of lung cancer cells. Vascular endothelial growth factor(VEGF) is an important factor regulating angiogenesis of non-small cell lung cancer(NSCLC); however,its pathologic features and significance are unclear. In this study,the tissue VEGF expression levels and its gene transcriptional status,as well as circulating VEGF levels,were investigated in patients with lung disease. Methods VEGF protein and m RNA expression levels in 38 lung tissue samples were analyzed by immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR),respectively. Circulating VEGF levels were detected quantitatively by an enzyme linked immuno-sorbent assay. Results The level of VEGF expression was significantly higher in lung cancer tissue than in the corresponding paracancerous or non-cancerous tissues. The average level of VEGF-positive staining was 76% in tissue samples from NSCLC patients; the levels were 89% in tissue samples from stage III patients and 92% in stage IV patients. High VEGF expression was also evident in cases with lymph node metastasis(84%),distant metastasis(90%),and lower differentiation degree(89%). VEGF m RNA in cancerous tissues was represented predominantly by the VEGF121 and VEGF165 isoforms. Circulating VEGF levels were significantly higher in NSCLC patients [(840 ± 324) pg/m L] than in patients with benign lung diseases [(308 ± 96) pg/m L] or in healthy individuals serving as controls [(252 ± 108) pg/m L]. Conclusion The over-expression of lung VEGF and its gene transcription status should be useful molecular indicators for NSCLC diagnosis.

Expression of PKD2 gene in human renal tissue and other tissues

Objective: To study the expression of PKD2 gene in human kidney and other tissues. Methods: The expression of PKD2 was detected by reverse transcription PCR(RT-PCR) and in situ hybridization(ISH) . The results of ISH were analyzed by micromegakargooytes. Results: Distribution of pkd-2 in normal adult kidney was stronger in proximal convoluted tubule, Henle's loop ascending branch, distal convoluted tubule and cortical collecting ducts, and inferior signal were observed in fetal kidney. Negative was seen in ADPKD 2 kidney. Conclusion: Down-regulation of PKD2 gene expression in kidney may take effect on the occurrence and development of ADPKD2.

Stem cell properties and neural differentiation of sheep amniotic epithelial cells

This study was designed to verify the stem cell properties of sheep amniotic epithelial cells and their capacity for neural differentiation. Immunofluorescence microscopy and reverse transcription-PCR revealed that the sheep amniotic epithelial cells were positive for the embryonic stem cell marker proteins SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and the totipotency-associated genes Oct-4, Sox-2 and Rex-1, but negative for Nanog. Amniotic epithelial cells expressed β-Ⅲ-tubulin, glial fibrillary acidic protein, nestin and microtubule-associated protein-2 at 28 days after induction with serum-free neurobasal-A medium containing B-27. Thus, sheep amniotic epithelial cells could differentiate into neurons expressing β-Ⅲ-tubulin and microtubule-associated protein-2, and glial-like cells expressing glial fibrillary acidic protein, under specific conditions.

EXPRESSION AND SIGNIFICANCE OF PTEN IN ENDOMETRIAL CARCINOMA

Objective: To investigate the expression of PTEN in endometrial carcinoma and its clinical significance. Methods: Reverse transcriptase-polymerase chain reaction and Western-blot methods were used to detect PTEN expression in 28 cases of endometrial carcinoma. Results: mRNA and protein expression levels of PTEN in endometrial carcinomas were significantly lower than those in normal endometrium (P<0.01). Conclusion: PTEN may play an important role in the tumorigenesis of endometrial carcinoma.

Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display

mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers （20 sets in total） were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ESTs were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ESTs were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ESTs were prepared for selecting genes related to adipose cells.

Sequence Analysis and Quantitative Detection of Norwalk-like Viruses in Cultured Oysters of China

We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） region of NLVs genomes with RT-PCR, the open reading frame 1 （ORF 1） of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts （copies） of NLVs in positive patient＇s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.

Predictive biomarker and clinicopathological characteristics analysis for esophageal cancer

Objective:The aim of this study was to explore the expression of manganese superoxide dismutase(MnSOD) in esophageal squamous cell carcinoma and its relationship with clinicopathological characteristics and its biological behavior.Methods:Immunohistochemical method(SP method),reverse transcription-polymerase chain reaction(RT-PCR) and Western blot were combined to detect the MnSOD protein and mRNA expression in 45 cases of esophageal squamous cell carcinoma and the normal tissue that was 5 cm apart from the edge of esophageal cancer lesion and without documented microscopic invasive cancer.Meanwhile,analysis was performed on the relationship between the pathological features of esophageal cancer and its biological behavior.Results:In esophageal squamous cell carcinoma and normal esophageal tissue,MnSOD protein expression was identified in 31.1%(14/45) and 86.7%(31/45)(P = 0.003),respectively,with the relative expression levels of MnSOD mRNA were 0.310 ± 0.036 and 0.482 ± 0.053(P = 0.000).The longer the lesions and the deeper the invasion,the differentiation would become poorer and the expression level of MnSOD would get lower,indicating that the level of MnSOD protein and mRNA expression were closely related to esophageal squamous cell carcinoma in the length of lesion,depth of invasion,and degree of differentiation(P < 0.05).Nevertheless,it showed no association with the presence of the lymph node metastasis,lesion site and the macroscopic classification(P > 0.05).Conclusion:The MnSOD protein and mRNA expression were both decreased in esophageal squamous cell carcinoma tissue.This may be related to the carcinogenesis and development of esophageal cancer.Detection of the expression of MnSOD would be of clinical significance in understanding the prognosis and guiding therapeutic strategy of esophageal cancer.