Effects of miRNA-21 on paclitaxel-resistance in human breast cancer cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of miR-21 on paclitaxel-resistance in human breast cancer MCF-7/PR and SKBR-3/PR cells.</p><p><b>METHODS</b>Paclitaxel-resistant human breast cancer cell lines MCF-7/PR and SKBR-3/PR were established by stepwise selection in increasing concentration of paclitaxel. Cellular morphology, mRNA and protein level of MDR1, BCRP and MRP1 in MCF-7/PR and SKBR-3/PR cells were determined. The expression of Bax, Bcl-2 and miR-21 in parental and paclitaxel-resistant cells was detected by RT-PCR and Western blotting. The synthetic miR-21 inhibitor or miR-21 mimic were transfected into MCF-7/PR, SKBR-3/PR and MCF-7, SKBR-3 cells with Lipofectamine 2000. The miR-21 levels were determined by RT-PCR, and P-gp, Bcl-2 and Bax protein levels were examined by Western blotting. MTT assay was used to measure the cell viability, and flow cytometry was performed to analyze the cell cycle and apoptosis.</p><p><b>RESULTS</b>The levels of MDR1, BCRP, MRP1, Bcl-2/Bax and miR-21 in MCF-7/PR and SKBR-3/PR cells were significantly higher than those in MCF-7 and SKBR-3 cells. The protein levels of P-gp, Bcl-2 were up-regulated, and Bax was down-regulated compared with parental cells. MiR-21 was significantly down-regulated after miR-21 inhibitor was transfected; and the levels of MDR1, BCRP, MRP1 and Bcl-2/Bax (P <0.05) were also down-regulated. MiR-21 inhibitors significantly suppressed G0/G1 transition of the cell cycle, and induced cell apoptosis in MCF-7/PR and SKBR-3/PR cells. MTT results showed that miR-21 inhibitors induced sensitivity of MCF-7/PR and SKBR-3/PR cells to paclitaxel. And miR-21 mimic can increase the expression of MDR1, Bcl-2/Bax and change cell morphology from parental cells to resistant cells.</p><p><b>RESULTS</b>The established MCF-7/PR and SKBR-3/PR breast cancer cells show typical multidrug resistance characteristics, which can be used as the model for drug resistance study. Down-regulated miR-21 expression in MCF-7/PR and SKBR-3/PR breast cancer cells can enhance cell sensitivity to paclitaxel.</p>

Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.</p><p><b>METHODS</b>Total RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.</p><p><b>CONCLUSION</b>This strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.</p>

Influence of recombination abnormality in spermatocyte meiosis on the pathology of patients with non-obstructive azoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To primarily study the influence of recombination abnormality in human spermatocyte meiosis on the pathology of the patients with non-obstructive azoospermia (NOA).</p><p><b>METHODS</b>We obtained testis tissues from 6 NOA patients by testicular biopsy and divided the tissue of each patient into 2 portions, one for pathological examination and the other for immunofluorescent staining. We observed the synaptonemal complex and the numbers of the recombination sites on homologous chromosomes, and analyzed the relationship between abnormal recombination and pathological findings.</p><p><b>RESULTS</b>Pathological examination showed that the basement membrane of the seminiferous tubules was thickened in 3 of the cases and atrophied in the other 3, the number of autosomal MLH1 foci in a spermatocyte ranging from 10 to 50 in the former 3, and from 30 to 50 in the latter 3.</p><p><b>CONCLUSION</b>The increased range of the homologous chromosomal recombination frequency may be one of the possible factors for the thickening of seminiferous tubule basement membrane and even lumen occlusion in NOA patients.</p>

Construction of pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and their interaction in living cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells.</p><p><b>METHODS</b>DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay.</p><p><b>RESULTS</b>The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.</p>

Abnormalities of meiotic recombination in Han Chinese azoospermic patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze defective homologous chromosomal recombination in Han Chinese azoospermic patients.</p><p><b>METHODS</b>Testicular biopsy samples from 7 healthy controls and 7 Han Chinese azoospermic patients including 2 obstructive azoospermia (OA group) and 5 non-obstructive azoospermia (NOA group) were analyzed. Immunofluorescence staining was performed to categorize early stage cells at meiosis prophase and to analyze chromosome pairing and recombination of pachytene spermatocyte. Newly developed meiotic proteins antibodies (anti-SCP3, anti-synaptonemal complex proteins 3, anti-MLH1, anti-Mut-L Homolog 1, anti-CREST, chromosome centromere antibody) were used to identify synaptonemal complex (anti-SCP3), recombination sites (anti-MLH1) and centromere (anti-CREST), respectively. Staging of spermatocyte was determined according to SCP3 formation progression. Qualitative data were compared by a Chi-square test, and ANOVA was used to analyze quantitative data.</p><p><b>RESULTS</b>Respectively, 2346 and 2932 spermatocytes were categorized in the controls and azoospermic patients. The proportions of zygotene cells in both OA group and NOA group were significantly higher than that of the control group. Investigation of 1967 pachytene cells from the controls and 354 pachytene cells from azoospermic patients indicated that the mean MLH1 foci per pachytene cell of NOA group was statistically lower than that of the controls. Compared with the controls, incomplete synaptonemal complexes cells (containing gap and/or split) were significantly increased in the NOA group.</p><p><b>CONCLUSION</b>Delayed meiosis prophase is relatively common in azoospermic patients, and changes in quantity and distribution of recombination foci may be the cause for spermatogenesis arrest in Han Chinese population.</p>

Effect of interfering hepatocyte nuclear factor-1 alfa in HepG2 on the expressions of apoM, apoA-I and the correlative key enzyme of cholesterol metabolism / 中华肝脏病杂志

To determine wether there were connections among hepatocyte nuclear factor-1 alfa (HNF-1a), liver receptor homolog-1 (LRH-1), apolipoprotein M (apoM) and to investigate the effects of HNF-1a in HepG2 on the expressions of apoM, apolipoprotein A-I (apoA-I) and the key enzymes in cholesterol metabolism and biotransformation. The mRNA expressions of apoM, LRH-1 and HNF-1a were detected by RT-PCR. HNF-1a was interfered and RT-PCR was used to detect the changes of apo M, apo A-I, Cyp7A1, farnesoid X receptor (FXR) and small heterodimer partner-1 (SHP-1). Western blot was used to detect the change of apo M protein. The expressions of apoM, LRH-1 and HNF-1amRNA were obviously higher in HCC tissue than that in para-cancer tissue (the vaule of t is -7.167, -7.075, -8.803, P less than 0.01 respectively). HNF-1a and LRH-1 positively correlated with the expression of apoM (r=0.353, P less than 0.01; r=0.523, P less than 0.01 respectively); RT-PCR and western blot results showed that the expressions of apoM, FXR and SHP-1 mRNA, could be obviously suppressed by HNF-1a interfering as compared to the negative controls by 47.4%, 47.9%and 65.2% (P less than 0.01) respectively, and the expression of apoM protein also decreased by 54.3% (F = 43.482, P less than 0.01). The expressions of HMGCR and CYP7A mRNA increased by 101.1% and 138.5% (P less than 0.01) respectively as compared to the negative control. But there is no effect on expression of apoA-I mRNA (F = 0.170, P more than 0.05). HNF-1a could promote cholesterol biotransformation by increasing the expression of apoM and the key enzymes in cholesterol metabolism and decreasing inhibiting factor. So HNF-1a provided protection against cardiovascular disease.

Colonic dripping with Taihuang liquid for treatment of neonatal hyperbilirubinemia / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the clinical effects of colonic dripping with Taihuang liquid (THL) in treating neonatal hyperbilirubinemia (HBE).</p><p><b>METHODS</b>One hundred and thirty-eight neonates with HBE were randomly assigned to two groups. Conventional treatment and nursing were given to both groups, and THL was given additionally to the observation group by colonic dripping.</p><p><b>RESULTS</b>Significant differences between the observation group and the control group were shown in frequency of defecation (4.6 +/- 1.3 times/d vs 2.0 +/- 1.1 times/d), daily serum bilirubin reduction (31.5 +/- 10.1 micromol/L vs 23.3 +/- 8.3 micromol/L), and days for normalizing serum bilirubin level (5.6 +/- 3.5 d vs 7.8 +/- 4.1 d, all P < 0.01).</p><p><b>CONCLUSION</b>Colonic dripping of THL could promote the excretion of bilirubin, so as to decrease the level of serum bilirubin in neonates with HBE.</p>

Characteristic Research on the Affinity of vMIP to Combine the Chemokine Receptors on Cell Membrane / 上海第二医科大学学报

Objective To clarify the combined ability of viral macrophage inflammatory protein (vMIP) to receptors through comparison research of vMIP binding to chemokine receptors of peripheral blood macrophages(PBMCs). Methods Combined ability of vMIP to receptors was measured with radioligand assay and identified with saturation, kinetic and specificity analyses. Results Kd is 11.1 nmol/L to human receptors of PBMC and 14.3 nmol/L to monkey receptors. The IC50 is 3.4 nmol/L to CCR5 and 4.5 nmol/L to CXCR4. Conclusion vMIP has high affinity with receptors and high specificity to CCR5 and CXCR4.This may be of great significance in the prevention and treatment of inflammation and HIV infection.