ObjectiveTo quantitatively determine the levels of type Ⅰ and type Ⅱ collagen mRNA in the intervertebral disc cartilage endplate of injured animal model, and to clarify the cytological function of intervertebral disc chondrocytes during fibrosis repair after intervertebral disc injury.MethodsForty healthy New Zealand rabbits were randomly divided into fibrosus puncture group, upper cartilage endplate single puncture group, upper and lower cartilage endplate multiple puncture group, and sham group. Two experimental animals were randomly selected from each group on the 2nd day, and the 2nd, 8th, 12th, and 24th week after the animal modeling operation to obtain intervertebral disc specimens. The levels of type Ⅰ collagen and type Ⅱ collagen in cartilage endplate cells of the intervertebral disc were determined by reverse transcriptase polymerase chain reaction (PCR). RNA was extracted from the endplate of the intervertebral disc, and the RNA concentration and the ratio of RNA concentration to protein concentration were determined by nucleic acid analyzer. Reverse transcription was performed by Revertaid M-Mulv reverse transcriptase, type Ⅰ and type Ⅱ collagen primers were designed to establish a PCR reaction system, 2% agarose gel electrophoresis (120 V, 40 min) was prepared by using 0.5×TBE electrophoresis buffer. The amplification results were observed under ultraviolet light, and the gray values of different electrophoresis bands were determined.ResultsThe level of type Ⅰ collagen mRNA in each operation group showed a progressive increase after 8 weeks, and the magnitude of the increase was related to the degree of injury. The level of type Ⅱ collagen mRNA showed a transient increase in the fibrosis puncture group and the upper endplate single-puncture group in the first two weeks after the endplate punctures were completed, and then began to decline progressively; in the multiple puncture group, it showed a downward trend from the beginning of the operation. ConclusionsThe synthesis of type Ⅰ collagen in chondrocytes of the injured nucleus pulposus tissue continues to increase with time, while the synthesis of type Ⅱ collagen begin to decrease progressively after a small increase. The formation and change of type Ⅰ and type Ⅱ collagen in injured intervertebral disc chondrocytes are different from natural degeneration.
Objective To evaluate the expressive varieties of Nogo-A mRNA in injured optic nerves of rats. Methods Reverse transcription polymerase chain reaction (RT-PCR) method was used to hemi-quantitatively analyze the levels of Nogo-A mRNA in the optic nerves 3, 7, 9, 15, 21, and 25 days respectively after injury.Results The level of the expression of Nogo-A mRNA was low in the normal optic nerves, while it was significantly high in the optic nerves 3 days after in jury, and kept the high level still after 25 days.Conclusion The expression of Nogo-A mRNA in injured optic nerves is increased. (Chin J Ocul Fundus Dis，2003，19：201-268)
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 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.
【Abstract】Objective To compare the sensitivity of HE,immunohistochemistry (IHC) and RT-PCR in detection of breast cancer metastases in axillary lymph nodes.MethodsTwenty female patients with newly diagnosed and clinically nodenegative breast cancers underwent modified radical mastectomy, including a complete axillary lymph node dissection. The ages of the patients ranged from 31 years to 65 years, and the diagnosis of breast cancer was approved by pathological finding. Two hundred and thirty-nine axillary lymph nodes were found in these 20 patients. Metastases in axillary lymph nodes were explored by HE， cytokeratin 19 IHC and RT-PCR for cytokeratin 19 respectively. ResultsSeven(2.9%) lymph nodes were found to have metastatic cancers by HE in 3 patients,all nodes were found in level Ⅰ. Metastatic cancers were found in 13(5.4%) nodes by IHC in 7 patients,11 nodes in level Ⅰ and 2 nodes in level Ⅱ; and 52(21.8%) nodes were found to contain tumor cells by RT-PCR in 14 patients,30 nodes in level Ⅰ and 22 nodes in level Ⅱ. All of 7 histologically(HE) positive nodes were found to contain tumor cells by IHC and RT-PCR. Among 232 histologically(HE) negative nodes,6(2.6%) nodes were found to contain tumor cells by IHC,and 45(19.4%) nodes were found to contain tumor cells by RT-PCR, all 6 IHC positive nodes showed the expected 460-base pair products on gel electrophoresis (P＜0.05).ConclusionThis study suggests that IHC and RT-PCR are more sensitive methods for the detection of micrometastases of breast cancer in lymph nodes than HE is,and RT-PCR is even better than IHC; the micrometastases of breast cancer in axillary lymph nodes could be detected accurately through these techniques.
【 Abstract 】 Objective To study the mRNA expression of BC047440 gene in multiplicate malignant tumor tissues and the corresponding adjacent tissues, and to investigate its roles in the carcinogenesis and development of malignant tumors. Methods Forty-eight cases of malignant tumor tissues and their adjacent non-cancerous tissues were examined. The mRNA expression of BC047440 gene in those tissues of liver cancer, cholangiocarcinoma, gastric cancer, carcinoma of large intestine, glioma, and breast cancer were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results ① The mRNA expressions of BC047440 gene in liver cancer, gastric cancer, cholangiocarcinoma and carcinoma of large intestine were significantly higher than those in their adjacent non-cancerous tissues (Plt;0.05 or 0.01). BC047440 gene were highly expressed in both glioma and its adjacent tissues (Pgt;0.05), and poorly expressed in both breast cancer and its adjacent tissues (Pgt;0.05). ② There were close relationships between BC047440 gene expression and clinicopathologic findings of liver cancer, including tumor size and portal vein invasion (Plt;0.05). ③ There were also close relationships between BC047440 gene expression and different clinical stages in alimentary canal cancers (Plt;0.05). Conclusion The over expression of BC047440 gene may be related with the growth, infiltration and metastasis of some malignant tumors, including liver cancer, cholangiocarcinoma, gastric cancer, carcinoma of large intestine and glioma.
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 detect the expression of forkhead box P3 （FOXP3 ）gene in esophageal squamous cell carcinoma(ESCC) and provide a new basis for immunotherapy of esophageal cancer. Methods Based on fluorescent TaqMan methodology, a realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting the expression of FOXP3 was set up. In this method, a cloning vector pMD 18-T-FOXP3 was constructed as a standard plasmid. The specific expression of FOXP3 in 42 patients with ESCC and 30 healthy controls were measured by using GeneAmp 7500 Sequence Detection Systems. Results FOXP3 mRNA copy number in ESCC was significantly higher than that in healthy control tissue ［(72.20±23.10)×104copy/μg RNA vs.(0.68±0.34)×104 copy/μg RNA；Plt;0.05］. Conclusion A realtime quantitative RT-PCR method for detecting the expression of FOXP3 gene in ESCC has been successfully established. The expression level of FOXP3 is increased in ESCC compare with healthy controls.
Objective To discuss the changes of c-kit/scf mRNA and protein in guinea pig gallbladder fed on high cholesterol diet. Methods Twenty guinea pigs were divided into two equal groups of 10 each：the control group and lithogenic group. Normal diet and high cholesterol diet was given to each group respectively. The period of stone permeation was six weeks. RT-PCR and Western blot were used to determin the expressions of c-kit and scf mRNA and protein. Results RT-PCR results showed that the expressions of c-kit mRNA（t=6.985，P＜0.01） and scf mRNA （t=6.028， P＜0.01）decreased significantly in lithogenic group compared with the control group. Western blot results showed that the expressions of c-kit protein （t=10.256， P＜0.01） and scf protein （t=9.586， P＜0.01）decreased significantly in lithogenic group compared with the control group. Conclusions The expressions of c-kit/scf mRNA and protein decrease during the formation of cholesterol gallstones in guinea pigs fed on high cholesterol diet. Inhibition of c-kit/scf pathway may play a role in the formation of cholesterol gallstones.