Objective To verify whetheriris pigment epithelial cells(IPECs)possess the similar potential of specific phagocytosis to retinal outer segments(ROS) with retinal pigment epithelialcells(RPECs). Methods IPESc were isolated from neonatal bovines with Hu's method,and were cultured.The cultured cells were identified by immunohistochemical methods with antibodies to cytokeratin and s-100.Total RNA of IPECs was extracted by Trizol.The specific primers for mannose-receptor andbeta;-actin were designed according to their sequence from Genbank.The mRNA expression of these proteins in the IPECs was analyzed by reverse transcription polymerase chain-reaction (RT-PCT).Results The Cultured IPECs have no contamination of other cells .The extracted RNA was ideal and had no degradation.RT-PCR analysis showed that mannose-receptor's mRNA was expressed in cultured IPECs in vitro.ConclusionCultured IPECs may express the mannose-receptor,and may have similar potential of phagocytosis to ROS with REPCs.
Objective To evaluate branched-chain DNA (b-DNA) signal amplification and semi-quantitative (Sq) RT-PCR in detection of free cancer cells in peritoneal flushing fluid of colorectal cancer patients during surgery. Methods The CEA mRNA in peritoneal flushing fluid in 48 cases of colorectal cancer were detected by b-DNA and SqRT-PCR. Peritoneal flushing fluid cytology (PLC) was conformed simultaneously to detect the free cancer cells. The peritoneal flushing fluid of 12 cases with colorectal benign disease were taken as negative control, GAPDH mRNA as internal control. Results In colorectal cancer patients, positive rate of free cancer cells by bDNA and SqRT-PCR （43.8%, 31.3%） was higher than that by PLC （4.2%）. The relative quantitative expressions of CEA mRNA were related to the Dukes staging， depth invasion and differentiation degree (Plt;0.05)， but irrelevant to tumor size，the patients’ age and gender (Pgt;0.05).Conclusion Both b-DNA and SqRT-PCR technologies have advantages and disadvantages to detect free cancer cells in peritoneal flushing fluid， which are related to clinicopathological factors.
Objective
To investigate the effect of hypoxia on expressions of erythropoietin（EPO）mRNA and protein in retinal Muuml;ller cells cultured in vitro.
Methods
Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.Muuml;ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase（RT）-polymerase chain reaction（PCR）and immunocytochemical method.
Results
Retinal Muuml;ller cells were cultured successfully，95% of which were positively stained by glial fibrillary acidic protein（GFAP）.Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal Muuml;ller cells，but increased obviously and time-dependently after hypoxia.
Conclusion
Expression of EPO mRNA and protein increases in Muuml;ller cells after hypoxia，which may be one of the protective factors for the nerves in anoxic retinopathy.
(Chin J Ocul Fundus Dis, 2006, 22: 196-199)
【Abstract】Objective To explore the changes of expression of AFP mRNA in human hepatocellular carcinoma （HCC） tissues after oral Xeloda therapy.Methods Total RNA was extracted from HCC tissue samples collect after operation and nested reverse transcription polymerase chain reaction （RT-nested PCR） assay was performed to determine the expression of AFP mRNA in this study.Results The final product of AFP mRNA amplified by RT-PCR was 174 bp and by RT-nested PCR was 101 bp. The AFP mRNA is positive in 12 of 21 patients (positive rate 57.14%) amplified by RT-nested PCR assay in Xeloda treatment group which is much lower than control group: 18 of 20 patients (positive rate 90.00%),P＜0.05.The serum AFP value of Xeloda treatment group 〔(23.2±12.8) μg/L〕 is much lower than that of control group 〔(39.6±24.3) μg/L〕 four weeks after operation (P＜0.05). However, There was no difference between two groups in serum AFP value before operation.Conclusion Xeloda can effectively suppress the expression of AFP mRNA in human HCC tissues and lower it’s product serum AFP value.The clinical application of Xeloda in HCC patients deserve further study.
ObjectiveTo evaluate the possible role of the expression of insulin-like growth factor-1 receptor (IGF-1R) in determining rectal cancer radiosensitivity. MethodsThe paired preradiation biopsy specimens and postoperative specimens were obtained from 87 patients with rectal cancer in the department of digestive tumor surgery, Jiangsu Province Hospital of Traditional Chinese Medicine, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine from January 2009 to December 2010. The IGF-1R expression was examined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). The tumor radiosensitivity was defined according to Rectal Cancer Regression Grade, then the relation between the IGF-1R expression and tumor radiosensitivity was evaluated. ResultsCompared with the preradiation biopsy specimens, IGF-1R expression significantly increased in the paired postoperative specimens of the residual cancer cells (Plt;0.001). The IHC result demonstrated IGF-1R overexpression was significantly associated with a poor response to radiotherapy (rs=0.401, Plt;0.001); RT-PCR detection of IGF-1R expression on preradiation biopsy specimens also showed that IGF-1R mRNA negative patients had a higher radiation sensitivity (rs=0.497, Plt;0.001). ConclusionDetection of IGF-1R expression may predict radiosensitivity of preoperative irradiation for rectal cancer.
Objective To identify and isolate the variant gene associated with gastric adenocarcinoma and clone the fragment of variant gene.Methods By arbitrarily primer polymerase chain reaction (AP-PCR), DNA samples from 5 matched gastric adenocarcinoma and non-tumor gastric tissues were analysed. Results The produced AP-PCR profiles were different in each matched gastric adenocarcinoma and non-tumor gastric tissue. One differentiated amplified DNA fragments PW2.2 from a matched gastric adenocarcinoma were cloned. The result of Southern blot hybridization with PW2.2 as a probe showing that this fragment was also found in some other gastric adenocarcinoma samples. Conclusion AP-PCR fingerprinting assay can be used to identify and clone the variant genes associated with gastric adenocarcinoma.
Objective To observe the effect of gefitinib on expression of epidermal growth factor receptor (EGFR) in bile duct epithelial cells, and the feasibility of inhibiting hyperplasia of bile duct epithelial cells with gefitinib. Methods Sixty-one patients with hepatolithiasis having to be in hospital for surgery from the First People’s Hospital of Shuangliu county were selected, with 25-65 years old, average 46.92 years. The patients were randomly divided into therapy group and control group. There were 30 cases in therapy group, in which fine duct was placed on lesion bile duct during operation, and through whom gefitinib solution was perfused after operation. There were 31 cases in control group with only T tube drainage after operation. The bile duct sample was obtained respectively during the operation and 6 weeks and 12 weeks after operation. The histology and expression change of EGFR were observed by HE staining, immunohistochemistry and RT-PCR method respectively. Results There were no significant differences in pathohistology changes of bile duct and the EGFR protein and mRNA expression between therapy group and control group during operation. The hyperplasia of epithelium mucosae and submucosal gland in the therapy group were obviously decreased as compared with those in control group, the EGFR mRNA and protein expression in therapy group were weaker than those of control group （Plt;0.05） 6 weeks and 12 weeks after gefitinib treatment. Conclusion EGFR is overexpressed in the chronic proliferative cholangitis, and continuously local application of gefitinib after operation can specifically interrupt the activation and expression of EFGR and then effectively inhibit the hyperplasia of bile duct epithelial cells.
【Abstract】 Objective To investigate the expression and significance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic cancer. Methods Thirty-two samples of pancreatic cancer tissue were collected from year 2002 to 2004. All of them were verified by histopathology and there were 9 cases of well-differentiated, 12 of moderately differentiated, and 11 of poorly differentiated, in which 12 cases were in the stage of Ⅰor Ⅱand 20 in the stage of Ⅲ or Ⅳ according to the TNM staging method. Eighteen normal pancreatic tissues were used as control group. The expressions of TRAIL receptors (death receptor 4, death receptor 5, decoy receptor 4 and decoy receptor 5) mRNA were assayed by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in the pancreatic cancer tissues and the normal pancreatic tissues. Results The expressions of death receptor 4 (DR4) and death receptor 5 (DR5) were detected in all the pancreatic cancer tissues and the normal pancreatic tissues and the levels of DR4 and DR5 were significantly higher than those of the normal pancreatic tissues (P＜0.01). Decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) were also expressed in normal pancreatic tissues, whereas DcR1 and DcR2 were only expressed in 18 and 20 pancreatic cancer tissues, respectively. However, there were no significant difference of the expression of DcR1 and DcR2 between the pancreatic cancer tissues and the normal pancreatic tissues （Pgt;0.05）. The expression level of DR5 in pancreatic cancer tissue was correlated with tumor differentiation and clinical stage, and the levels in stage Ⅲ and stage Ⅳwere significantly lower than those of stageⅠand stageⅡ(P＜0.05). The expressions of DR4, DcR1 and DcR2 were not correlated with tumor differentiation and clinical stage (Pgt;0.05). Conclusion ①The expression of TRAIL receptors in pancreatic cancer tissues is prevalent, but the types of receptors expressed in different tissues were also different. High expression of death receptors may play an important role in TRAIL recptors regulated pancreatic cancer apoptosis. ②The expression of DR5 is correlated with the differentiation degree of pancreatic cancer cell and clinical stage of tumor. The expressions of DR4, DcR1 and DcR2 should not be considered as related indexes of differentiation degree or clinical stage of pancreatic cancer.
Objective To investigate the transfection and expression of recombinant plasmid human vascular endothelial growth factor 165/pcDNA3. 1 （hVEGF165/pcDNA3. 1） in myocardial cells, and to build foundation for gene therapy and cell therapy of coronary artery disease （CAD）. Methods Myocardial cells were cultured in vitro and transfected by hVEGF165/pcDNA3.1 with liposome; then transient expressed protein was detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, immunochemistry and Western blotting. Results A strap as hVEGF165 was obtained by RT-PCR, the protein of hVEGF165 was found in myocardial cells by immunochemistry and in supernatant by Western blotting. Conclusion The recombinant plasmid hVEGFI65/pcDNA3. 1 can be expressed in myocardial cells, and may be used in studying CAD by gene therapy and cell transplantation.
【Abstract】ObjectiveTo discuss the possibility and clinical significance of SSX-1 mRNA used as specific marker for examining HCC cells in peripheral blood of HCC patients. MethodsUsing the RT-PCR method, the SSX1 mRNA of the peripheral blood was examined in 25 cases of HCC patients and 20 non-HCC patients. The same method was used to detect the expression of SSX-1 mRNA in the tumor tissues, para-tumor tissues, cirrhosis tissues and normal hepatic tissues. A randomized sample was extracted for DNA sequencing from positive electrophoresis expression samples of SSX-1 to examine the reliability of results. ResultsThe expression rates of SSX-1 mRNA were 60%(15/25) and 40%（10/25） respectively in tumor tissues of HCC and the corresponding peripheral blood. SSX-1 mRNA were not expressed in para-tumor tissues,cirrhosis tissues and normal hepatic tissues. The DNA sequence -confirmed that the RTPCR products were true target cDNA. No relationships were found between the expression of SSX-1 gene and clinical characteristics, such as age, sex, tumor size, TNM stage， extent of differentiation and serum AFP level (Pgt;0.05). However, in 33%（3/9） patients with normal serum AFP (lt;20 μg/L), specific expression of SSX1 mRNA was observed. ConclusionHigh specific expression of SSX-1 mRNA is observed in the peripheral blood of patients with HCC, it suggests that applying it as a tumor marker to detect HCC cells in peripheral blood is an adjuvant diagnostic tool. The expression of SSX-1 mRNA in the peripheral blood is observed in the group HCC patients whose serum AFP (lt;20 μg/L) are normal, which suggests that applying both SSX-1 mRNA and AFP as tumor markers together might be useful to improve the diagnostic accuracy for HCC.
