ABSTRACT
Objective To construct miR-223 knockdown lentivirus vector and provide a tool for further study of the function of miR-223. Methods According to the Invitrogen miR-RNAi online design tool, a pair of complementary oligo?nucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vec?tor. Then miR-RNAi expression cassette was cut and subcloned into lentiviral pCDH-CMV-MCS-EF1-copGFP vector. The lentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calcu?lated, and the knockdown of endogenous miR-223 was detected by using real-time RT-PCR. Results Restriction enzyme digestion and sequencing results showed that miR-223 lentivirus construct was successfully made. Lentivirus that knock?down miR-223 expression packaged and infected of target cells. The expression of GFP green fluorescent protein accounted for 80%-90%and the virus titer was 1×109 PFU/mL. The infection efficiency reached 90%. Compared with negative control virus, miR-223 knockdown lentivirus significantly down-regulated the expression of miR-223 in ST2, and was 31%(n=3, t=15.091, P<0.05). Conclusion miR-223 knockdown lentivirus is successfully made. It provides a tool for further studying the function of miR-223.
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Objective To explore the effect of applying Case-based study (CBS) in combina-tion with Reference-induced self education (RISE) in clinical teaching of Digestive endoscope diag-nostics. Method One hundred and twenty undergraduates in Grade 2012 of clinical imaging speciality of Beihua University were selected as the objects of study and classified at random into experiment group (60 students) and control group (60 students), In the course of teaching, the traditional LBL study and CBS-RISE study were separately employed, and examined through theoretical assessment in combination with case analysis, and the teaching effect in experiment group students was also evaluated through examination paper. SPSS 16.0 statistic soft ware was employed for data treatment and analysis, t test was used for quantity data and expressed as x±s, and x2 test for number counting data, and P<0.05 as the statistical significant difference. Results In experiment group, the total result, and the results of theoretical examination and case analysis were all superior to the control group, and the dif-ference between two groups exhibited statistical significance (P values respectively 0.008, 0.017 and 0.021). The excellent and good rate of the experimental group's theory examination score was 70%, which was higher than that of the control group (48.3%). The excellent and good rate of the experimen-tal group in the case analysis of examination results was 63.3%, higher than that of the control group (43.3%), and the difference was statistically significant (P values respectively 0.016 and 0.028). The students in experimental group showed better degree of satisfaction to the teaching model of CBS-RISE. In general, they considered that the CBS-RISE model could stimulate the learning interest of students, enhance the ability of analyzing and solving problems, deepen the understanding of knowl-edge, train a better clinical thinking model, and also develop a cooperative group idea. Conclusions CBS-RISE teaching model is feasible in the application of teaching in digestive endoscope diagnostics, and beneficial to the training of self learning ability and clinical consideration, deserving populariza-tion.
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Objective To investigate the expressions of apoptosis stimulating protein of P53 (ASPP)family in colorectal carcinoma tissue and to explore their relationship with the clinicopathological characteristics of colorectal carcinoma,and to clarify the effect of ASPP family in the development of colorectal carcinoma.Methods 45 cases of colorectal carcinoma tissue and 20 cases of healthy controls were selected. Among 45 cases of colorectal carcinoma tissue, there were 1 1 cases of well differentiated colorectal carcinoma, 2 1 cases of moderately differentiated colorectal carcinoma,and 13 cases of poorly differentiated colorectal carcinoma;7 cases of T1 stage colorectal carcinoma,8 cases of T2 stage colorectal carcinoma,25 cases of T3 stage colorectal carcinoma,and 5 cases of T4 stage colorectal carcinoma;19 cases of N1 stage with lymph node metastasis,26 cases of N0 stage without lymph node metatasis.The expressions of ASPP1,ASPP2,and iASPP in 45 cases of colorectal carcinoma tissue and 20 cases of normal colon tissue were detected by immunohitochemistry SP method,and the correlations between the expressions of ASPP family and the pathologic typing,infiltrative depth,and lymph node metastasis of colorectal carcinoma were analyzed. Results ①The immunohitochemical staining results showed that the ASPP family members expressed in colorectal carcinoma tissue and normal colon tissue, and there were no significant differences in ASPP1 and ASPP2 positive rates between colorectal carcinoma tissue and normal colon tissue (P>0.05);the positive expression rate of iASPP in colon cancer tissue was higher than that in normal colon tissue (P0.05);the expression of ASPP2 positive rate was decreased when the differentiation degree of tumor cells reduced,they had positive correlation (rs=0.454,P=0.002);the expression of iASPP in colon cancer tissue had no correlation with the differentiation degree of tumor cells (rs=-0.171,P>0.05).③ The expression of ASPP1 in colon caner tissue had no correlation with the infiltrative depth of tumor (rs=-0.268,P>0.05);the expression of ASPP2 positive rate was decreased when the tumor infiltrative depth increased,they had negative correlation (rs=-0.348,P0.05).④The expressions of ASPP1,ASPP2, and iASPP in colon caner tissue had no correlation with lymph node metastasis (rs=0.089,rs=0.044,rs=0.210, P>0.05).Conclusion The expression levels of iASPP in colon cancer and normal colon tissues are different,it may be useful for the diagnosis, differential diagnosis and evaluation in benign and malignant colorectal diseases. The expression of iASPP is negatively correlated with the pathologic typing and neoplasm staging of colorectal carcinoma,it indicates that iASPP can be used as a indicator in judging the prognosis of colorectal carcinoma.
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Objective To investigate the effect of 1, 25-dihydroxy-vitamin D3 (1, 25 (OH)2D3) on adipocyte differen-tiation and the underlying mechanism. Methods The mesenchymal stem cell line C3H10T1/2 was randomly divided into 6 groups including control group, differentiation group and 4 different doses of 1, 25(OH)2D3 groups. The control group was treated with vehicle. The differentiation group was supplemented with adipocyte differentiation reagent. And the 1,25(OH)2D3 groups were treated with adipocyte differentiation reagents and 10-9, 10-8, 10-7 and 10-6 mol/L of 25(OH)2D3. After culturing for 5 days, the cells were stained with oil red O, and the expression levels of adipocyte-specific transcription factors and Wnt/β-catenin signaling pathway related genes were examined by RT-PCR or Western blot methods. Results 1,25(OH)2D3 sig-nificantly reduced the number of differentiated adipocytes and blocked the mRNA levels of adipocyte specific transcription factor PPARγ(peroxisome proliferator-activated receptor gamma), C/EBPα(CCAAT enhancer binding proteinα) and adipo-cyte characterization factor aP2 (fatty acid binding protein 4). These were paralleled by the decreased mRNA expression of Wnt/β-catenin signaling pathway inhibitor sFRP1 (Secreted frizzled-related protein 1) and the increased level ofβ-catenin protein. Conclusion 1, 25(OH)2D3 inhibits adipocyte differentiation, which may be related to the activation of Wnt/β-catenin signaling.