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.
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 determine the characteristics and regularity of fibronectin mRNA expression in diabetic ulcers, and to investigate the relationship between the changes of fibronectin mRNA expression and pathogenesis of diabetic ulcer. METHODS Biopsies were removed from the margins of diabetic foot ulcers, included surrounding skin as experimental group, and the biopsies from normal skin of the same patients as control group. The mRNA expression of fibronectin was measured by quantitative RT-PCR technique. RESULTS The mRNA expression of fibronectin could be detected in both normal skin and diabetic foot ulcers, but the level of expression in diabetic ulcers was lower than that of normal skin. CONCLUSION The level of mRNA expression of fibronectin in diabetic ulcers is decreased, which suggest that the down-regulation of transcription may be one of the mechanisms of chronic impaired ulcers.
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)
Objective To investigate the detection of peritoneal free cancer cells and its clinical significance. Methods The peritoneal free cancer cells, the positive rates of CK20 protein and CK20 mRNA expressions of peritoneal lavage fluid were detected by peritoneal lavage cytology (PLC), flow cytometry (FCM) and real-time fluorescent quantitative RT-PCR in 50 cases of gastric cancer patients, respectively. The sensitivity of three kinds of detection method to peritoneal free cancer cells was compared. Results The positive rates of peritoneal free cancer cells, CK20 protein and mRNA expression of peritoneal lavage fluid were 20.0% (10/50), 36.0% (18/50) and 58.0% (29/50), respectively. The positive rate of CK20 mRNA expression detected by real-time fluorescencequantitative RT-PCR in peritoneal lavage fluid was significantly higher than those of the CK20 protein expression detected by FCM and peritoneal free cancer cells detected by PLC (Plt;0.05 or Plt;0.001). The difference of positive rate of CK20 protein expression and peritoneal free cancer cells was not significant (Pgt;0.05). The positive rate of CK20 mRNA expression of peritoneal lavage fluid was related to the tumor invasion depth, differentiation degree, TNM stage, and lymph node metastasis (Plt;0.05). Conclusion Real-time fluorescence quantitative RT-PCR is an effective method for the detection of peritoneal free cancer cells.
Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
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 investigate the reliability of culture method of adult human parathyroid cells. Methods Adult human parathyroid tissue was digested by collagenase, then the original generation of cells were cultured and passaged, and their morphological changes were observed and recorded every other day. Part of the passaged cells were observed through electron microscope and its supernatant parathyroid hormone (PTH) was assayed. Meanwhile, the other part of cells were tested the parathyroid markers, including PTH, calcium-sensing receptor (CaSR) and glial cells missing-2 (GCM-2) by PCR. Results Abundant cytoplasm, mitochondria, endoplasmic reticulum and Golgi apparatus from the seventh day's passaged cells were observed by the electron microscopy, as well as, some secretory granules existing in both cytoplasm and intercellular lacuna. Also, the PTH from supernate was detected, and parathyroid specific markers, such as CaSR, PTH, and GCM2 were positive. Conclusions These trials demonstrated the adult human parathyroid cells could be harvested by collagenase digestion and the cultured. Furthermore, the cells remained good shape and kept functioning, making it a potential source for allogeneic cell transplantation to the treatment of permanent hypoparathyroidism.
【Abstract】Objective To investigate whether liver resection for hepatocellular carcinoma (HCC) causes dissemination of liver tumor cells into blood circulation. Methods Fourteen patients with HCC, but without evidences of metastasis, were enrolled for the study. Blood samples of peripheral blood before skin incision and after abdominal wall suture, and of hepatic venous blood and portal venous blood after liver parenchyma dissection, were obtained. AFPmRNA was detected by reverse transcription polymerase chain reaction assays, the change of the level of its expression during operation was assessed by semi-quantitative analysis. Results The rate of its expression before and after operation in peripheral blood, and during operation in portal venous blood and in hepatic venous was 42.9%, 35.7%, 42.9% and 57.1% respectively. There were no differences between them. However, the level of its expression in hepatic venous blood was significantly higher than others (P＜0.05). Conclusion Liver resection for HCC induces releases of cells from the liver, probably including tumor cells, into blood circulation.
【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.