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Aim To screen a more suitable transfection recep-tor,and improve the efficiency of constructing cell lines highly expressing human peptide transporters 1 (hPepT1 ).Methods The recombinant plasmid pcDNA3.1 (+)-hPepT1 was transfect-ed into MDCK cells and HeLa cells by LipofectamineTM 2000 transfection reagent,respectively.The monoclonal cells were se-lected and cultured.Expression of hPepT1 mRNA and protein were determined by qRT-PCR and Western blot,respectively. The uptake capacity of Glysar in transfected cells was examined. Results Compared with wild type cells,the expression of hPepT1 and the uptake of Glysar in transfected MDCK cells and HeLa cells significantly increased (P <0.05).Although the up-take of Glysar in HeLa cells was higher than that of MDCK cells,on the contrary,the expression of hPepT1 and the uptake of Glysar in MDCK-hPepT1 cells was higher than that of HeLa-hPepT1 cells.Conclusion MDCK cells may serve as a more suitable transfected receptor for the construction of a cellular model with high expression of hPepT1 ,which would make the construction of a cell model highly expressing hPepT1 more effi-cient.
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Objective To evaluate the clinical value of real-time fluorescent quantitative reverse transcription polym ERαse chain reaction (qRT-PCR) in detecting expression of estrogen receptor alpha (.ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HerZ) genes in breast cancer tissues. Methods Totally 48 breast cancer tissues and 28 benign breast tumor tissues (control) were obtained from patients undergoing surgery in our hospital during Mar. 2010 and Oct. 2010. The expression of ERα, PR and Her2 protein was examined by immunohistochemistry (IHC) in breast cancer tissues and the expression levels of ERα, PR and HerZ mRNA were detected by real-time qRT-PCR in breast cancer tissues and benign breast tumor tissues. The values of these two methods in diagnosis of breast cancer were evaluated. Results The expressions of ERα and HerZ mRNA were significantly higher in the breast cancer tissues than in the controls (P0. 05). Real-time qRT-PCR in detecting ERα, PR and Her2 mRNA expression had similar capability with IHC method in evaluating the sensitivity, specificity of endocrine thERαpy; moreover, the two methods also had a consistent pathological diagnosis rates (P>0. 05). Conclusion ERα, PR and Her2 genes are important predictive markers for endocrine thERαpy or targeted thERαpy of breast cancer. Real-time qRT-PCR method can be used for clinical detection and research of ERα, PR and Her2 mRNA.
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Objective To investigate the expression and significance of intermedin (IMD) and its.receptors CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues of patients with non-small cell lung cancer.Methods The mRNA gene expressions of IMD,CRLR,RAMP1,RAMP2 and RAMP3 were detected by realtime quantitative RT-PCR in cancerous and para-cancerous tissues from 27 patients with lung cancer.Results Real-time quantitative PCR detection results showed that the expression of IMD,CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues were [(59±7.9)×10-8,(96±2.7)×10-6,(29±3.9)×10-9,(14±2.6)×10-6,(65±1.1)×10-6]which were higher than those in adjacent tissues[(40±4.7)×10-10,(21 ±3.9)×10-6,(53±7.8)×10-10,(64±1.9)×10-8,(36±1.3)×10-9] to some extent (all P ＜ 0.05); the higher expression of RAMP3 was found is higher expressions than RAMP1 and RAMP2 in cancer tissues (all P ＜ 0.05).Conclusion The expressions of IMD and its receptors in cancer tissues are higher than those in paracancerous tissues.IMD may play an important role in the development of cancer by activate RAMP3 which is the most high expressed receptor in cancer tissues.Therefore,it might be helpful for the investigation of new gene thereapy in non-small cell lung cancer.
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Objective To explore the clinical significance and the impact on prognosis of blood micrometastasis in the patients with pN0 esophageal squamous cell carcinoma. Methods Total RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA. RT-PCR was used to detect MMP-7 mRNA and hTERT mRNA in blood. △△Ct sample values were calculated with post-operative follow-up of 3 month, 6 month, 12 month. Results Statistical results suggested that blood micrometastasis was related to differentiation grade and pTNM staging (P=0.000, P=0.000 respectively), but not to age, sex, length of turnout (P0.05). Follow-up results suggested that the degree of invasion and tumor metastasis (recurrence) was no correlation; follow-up to 6 month and 12 month, tumor metastasis (recurrence) was associated with blood micrometastasis, and follow-up to 12 month, compared with the tumor metastasis (recurrence) probability of blood micrometastasis-positive patients and negative patients, the former was as 6.44 times as the latter. (OR=6.440, 95 % CI 1.547-26.822). Conclusion Blood micrometastasis testing is of great significance to early diagnosis and prognosis judgment in pN0 esophageal squamous cell carcinoma patients.
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Background and purpose:Conventional cytology is valuable in diagnosing the cancer cells in lymph nodes of patients with lung cancer. However , the diagnostic value of detecting lymph node and paracancer micrometastasis was limited. Our study was to investigate the mRNA expression level of lung specif ic X protein(LUNX) and cytokeratin 19(CK19) and its signifi cance in the carcinogenesis,metastasis and prognosis of NSCLC. Methods: Quantitative analysis with reverse transcriptase polymerase chain reaction(Real-Time RT-PCR) was used to detect the mRNA expression level of LUNX and CK19 in 20 tumor tissues and 42 regional lymph nodes from 20 patients with NSCLC, and 6 lymph nodes from 9 patients with pulmonary benign diseases as control. Meanwhile, all lymph nodes were also examined by conventional pathological method. Results:①the mRNA level of Lunxin in cancer Tissue was signifi cantly higher than that in the control group, but there was no association with Lymphatic Metastasis,Tumor Size and TNM grade.②the mRNA level of Lunx in the cancer Tissues was signif icantly higher than that in the control group, and was associated with Lymphatic Metastasis,Tumor Size and TNM grade.③the mRNA of CK19 in lymph nodes was almost the same as control, and not associated with tumor size,lymphatic metastasis and TNM grade.④the mRNA of LUNX in lymph nodes was almost the same as control, which has no acossiation with lymphatic metastasis,tumor size and TNM grade.⑤ mRNA of CK19 and LUNX was unrelated to age,sex and histological type .⑥there was signifi cant difference between using Real-Time RT-PCR methods and the routine pathological methods to detect lymph node metastasis in lung cancer. Conclusion:This result suggested that the detection of LUNX message RNA and CK19message RNA might be helpful to diagnose NSCLC micrometastasis in lymph nodes. LUNX was superior to CK19 both in sensitivity and specifi city. The establishment of this method may increase the positive rate of detecting metastatic regional lymph nodes in non small cell lung cancer.
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Objective:To develop a real time fluorescent quantitative reverse transcriptase polymerase chain reaction(RT-PCR) system for determining the expression of Dicer mRNA in human hepatoma cell lines and 20 samples of primary hepatocellular carcinoma(PHC)tissues.Methods: The specific primers,designed according to the complete sequence of Dicer mRNA,and the fluorescence dye SYBR Green Ⅰwere used for RT-PCR amplification.The fluorescence was monitored in a real time manner.The expression levels of Dicer mRNA in samples were calculated according to the standard curve and the nonspecific amplifications were excluded by melting curve analysis.The mRNA levels of Dicer were presented as the ratios of Dicer mRNA to 18S rRNA.Results: The linear detection range of this system was from 5?10~(2)to 5?10~(9)copies/?l and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 4.13% to 19.72% and from 6.25% to(18.76%,) respectively.The expression levels of Dicer mRNA in HBV positive hepatoma cell line HepG2.2.15 or in HBV negative hepatoma cell line HepG2 were significantly lower(P