Objective: To establish a doxorubicin resistant cell line A549/ADM and research the relationship between A549/ADM and human lung carcinoma stem cell. Methods:  Doxorubicin resistant lung adenocarcinoma cell line A549/ADM was obtained by treating lung adenocarcinoma A549 in vitro with increasing dosage of doxorubicin in serum free medium. The resistant to chemotherapeutic drugs was evaluated with MTT assay. Cell cycle, expressions of ABCB1 and ABCG2 and concentration of doxorubicin was determined by flow cytometry. CD133 positive cells explore were determined by the immunofluorescent staining and flow cytometry to examine lung carcinoma stem cell proportion. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Results: A549/ADM cells were proved multidrug resistant. The cells showed resistance to ADM，VCR，Taxol, and the resistant index of A549/ADM was 13.167，12.86，3.4. Cells in the period S was (45.76±1.41)% in A549 whereas (23.33±1.32)% in A549/ADM.The apoptosis of A549 cell was (58.23±0.82)% whereas (16.49±0.77)% in A549/ADM. Compared with the parental cell line, A549/ADM cell had a higher expression level of ABCB1 and ABCG2（P<0.05）.The concentration of doxorubicin in the A549/ADM is obviously lower than that in the A549 cell. The immunofluorescent staining and flow cytometry analysis showed that the expression levels of CD133 were significantly higher in A549/ADM cell. A549/ADM cell were highly tumorigenic in nude mice.  Conclusion:  Doxorubicin resistant lung adenocarcinoma cell line A549/ADM was obtained by treating lung adenocarcinoma A549 in vitro with increasing dosage of doxorubicin in serum free medium.cell line A549/ADM had a higher proportion of lung adenocarcinoma stem cells compared to its parental cell line A549.

Objective: To optimize the cultivation method of cerebral astrocytes from mice in vitro, in which develop an experimental basis for the functional study of astrocytus.  Methods: Cerebral cortex was obtained from 0 to 1d C57BL/6 mice and cell suspension was prepared by digestion with trypsin digestion and mechanical dissociation. After differential attachments, primary cells were cultivated for 24 h, so the fresh culture medium instead of the old was added softly. After then, the cell flask was washed shakely before the old culture medium was replaced with the fresh medium every two or three days. After three passages, astrocytus in the culture medium were stained with anti-glial fibrillary acidic protein (GFAP) antibody using immunocytochemical or immunofluorescence staining.  Results:  After primary cell culture and subculture, the purified astrocytes had the typical shapes with a purity beyond 95%.  Conclusion:  A reproducible and simple cultivation method in vitro of astrocytus was established, in which high purity astrocytes were obtained.

 

[Abstract]Objective: To study the microRNA(miRNA) targeting to 3′untranslated region(3′UTR) of suppressors of cytokine signaling3(SOCS3) gene, a SOCS3 3′UTR luciferase reporter vector was constructed and to analyse the miRNAs regulated SOCS3 expression through detecting the luciferase activity of SOCS3 3′UTR. Methods:  The 3′UTR fragment of SOCS3 gene was amplified by PCR from genomic DNA of primary astrocytes of mouse and cloned into luciferase reporter vector (pGL3Promoter), then the recombinant pGL3Promoter/SOCS3 was identified. The miRNAs targeting SOCS3 3′UTR was predicted by Target Scan5.2, RNAhybrid and FINDTAR3 softwares. Thereafter, pGL3Promoter/SOCS3 and miRNAs were cotransfected into HEK 293T cells and the luciferase activity of SOCS3 3′UTR was measured. Results: 3′UTR fragment of SOCS3 gene was successfully cloned into the pGL3Promoter reporter vector, which verified by Xba I digestion and DNA sequencing. The predicted miRNAs targeting SOCS3 3′UTR included miR203, miR291a5p, miR9, miR140 and miR130b. Compared with the control group, the luciferase activity of pGL3Promoter/SOCS3 treated with miR203, miR291a5p, or miR140 was remarkably decreased, respectively. Conclusion: The SOCS3 3′UTR luciferase reporter vector was constructed successfully, and the luciferase activity of the recombinant vector can be suppressed significantly by miR20, miR291a5p or miR140.

目的:  探讨镇江地区感染人乳头瘤病毒（human papillomavirus,HPV）的女性子宫颈病变情况及HPV基因型别分布特点。方法: 选择在19种HPV基因型感染筛查中HPV结果阳性的女性978名，进行阴道镜检查并活检，以病理结果作为诊断宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）的金标准。结果：  978名女性中无CIN病变、CINⅠ、CINⅡⅢ和宫颈癌的检出率分别为89.26%（873/978）、5.42%（53/978）、4.91%（48/978）和0.41%（4/978），CINⅠ、CINⅡⅢ和宫颈癌组的HPV基因型别分布有差异，随宫颈病变程度增高， 16和18型的HPV检出比例增加，宫颈高度病变患病高峰为36～40岁年龄段。978名妇女检出19种基因型HPV共1 240频次，其中高危型931例（75.08%），以HPV52、58、16、51、18和39型多见,低危型97例（7.82%），以HPV43型多见，中国人常见亚型共212例（17.10%），以HPV53、CP8304型多见；HPV单一型别感染为80.16%(784/978），多重型别混合感染为19.84%（194/978），多重感染率随年龄增长呈下降趋势。不同等级宫颈病变间的HPV多重感染率差异无统计学意义(P＞0.05)。结论： 镇江地区生殖道有HPV感染的女性子宫颈病变多处于发病早期，子宫颈病变严重程度与HPV多重感染无关，而与不同基因型HPV感染有关，HPV16/18型与宫颈病变进展关系密切。  

Objective: To establish an efficient method for the isolation of murine peritoneal macrophages by comparing the biological characteristics of mouse peritoneal macrophages with different methods. Methods: Thirtytwo healthy Kunming mice were selected and randomly divided into 4 groups, and macrophages were obtained by four different methods, i.e., PBS direct peritoneal lavage(PBS group), intraperitoneal injection of RPMI 1640 medium containing 10% serum for 30 min followed with PBS lavage(medium group), intraperitoneal injection of 6% starch broth for 3 d followed with PBS lavage(starch broth group) and intraperitoneal injection of 3% thioglycollate broth for 3 d followed with PBS lavage(thioglycollate group). The obtained cells were then transferred into the RPMI 1640 culture medium containing 10% serum and cultured. The cell morphology, number, phagocytic ability, purity and viability were compared. Results: There was no significant difference in the morphology of macrophages among the four groups; the cell number and the phagocytic ability in thioglycollate broth group were significantly higher than those of other three groups(t=16.685, 9.361, 3.199, all P0.05), which were greater than 95% respectively. Conclusion: Intraperitoneal injection of 3% thioglycollate broth for 3 d followed with PBS lavage, is a highly efficient method for obtaining mouse peritoneal macrophages.

Objective: To establish a new method to isolate and culture mouse primary pancreatic stellate cells(PSCs). Methods: Based on techniques of isolating and culturing rat PSCs, we acquired purified PSCs from pancreatic tissue fragments via isolation, culture and passage. Oil red O staining, immunofluorescence as well as western blotting were performed to identify pancreatic stellate cells. Cell growth and vitality were validated by cell counting and trypan blue staining. Results: Primary PSCs were positive for oil red O staining; cells grew rapidly and began being activated from the third day. After passage, most of cells were activated. Immunofluorescence staining showed that α-smooth muscle actin (α-SMA) was positive expressed in activated PSCs. Western blotting showed that α-SMA and desmin were expressed in activated PSCs, and that the expression of desmin in fully activated PSCs was obviously higher(P＜0.05).Conclusion: This method can be successfully used to isolate mouse PSCs. The yield, purity and activity of PSCs were higher, and this new method could thus meet the research of pancreatic stellate cells in vitro.

Objective: To investigate the relationship between p38 MAPK （or phosphorylation-p38 MAPK) levels in peripheral blood mononuclear cell (PBMC) and disease severity in patients with acute pancreatitis (AP). Methods:  Twenty three patients with severe acute pancreatitis (SAP), 29 patients with mild acute pancreatitis (MAP), and 21 healthy volunteers were evaluated in this study. Levels of p38 MAPK mRNA in PBMC were measured by real-time PCR. Protein levels of p38 MAPK and P-p38 MAPK were detected by Western Blot. The data obtained were subjected to one-way ANOVA. Results:  Levels of p38 MAPK mRNA in SAP group were increased by 11.7% (P<0.05) compared with control group, while there were no difference between MAP and control groups. Levels of p38 MAPK protein in SAP group were increased by 18.7% (P<0.05) compared with control group, while there were no difference between MAP and control groups. Phosphorylation levels of p38 MAPK in SAP group were significantly increased by 444.1% (P<0.01) contrast to control group, and increased by 44.1%（P<0.05）compared with MAP group. That in MAP group was elevated by 27.8%（P＜0.05）compared with control group. Conclusion:   It was phosphorylation levels but not total levels of p38 MAPK that significantly increased in PBMC from acute pancreatitis, and phosphorylation-p38 MAPK took a close relationship with the grade severity of AP.

Objective: To investigate whether hepatocytes could undergo epithelial-mesenchyme transition（EMT）on recombinant TGF-β1 stimulation in vitro . Methods: Human hepatocyte line HL7702 were cultured for 2 days with 20% NBS（1640）nutrient medium，and changed for serum-free 1640 medium and  TGF-β1 factor induced HL7702 cells for 24，48，72 h. Cells morphology changes were watched before and  after induction with inverted microscope. Real-time RT-PCR and western blot were used to analyze the expression of epithelial and mesenchyme cells-associated genes and proteins. Results: Hepatocytes before  TGF-β1 induction were irregular sixangle cells. After stimulation cells showed spread and spindle-shaped morphology and cell-cell junction got bigger. Real-time RT-PCR and western blot analysis showed that epithelial marker-associated E-cadherin expression decreased obviously after TGF-β1 induction，while mesenchyme markers N-cadherin、Vimentin、Twist increased significantly. Conclusion: Human hepatocyte line HL7702 happened to EMT by recombinant TGF-β1 stimulation. EMT associated epithelial and mesenchyme genes and proteins expression changed significantly. It could be served as a foundation for further investigation on the mechanism of hepatocytes EMT.

Objective: To establish a mice model of colitis associated cancer. Methods: The colitis associated cancer was induced by a single intraperitoneal injection of 12.5 mg/kg azoxymethane (AOM) and stepped by four cycles oral administration of 2% dextran sodium sulpate (DSS). The occult blood was observed everyday. At the end of the fouth cycle, the mice were killed for observing the spleen, mesenteric lymph nodes, colon; colon tissues were harvested for mucosa morphological changes under light microscopy. Results: Compared to the control group, the spleens, mesenteric lymph nodes were enlarged and the tumorigenesis was found in distal colon in the experiment group; and there were a lot of inflammation cell infiltration and dysplasia in colon. Conclusion: Administration of AOM combined with DSS could induce a mice model of colorectal cancer in a short period.

Objective: To optimize the method of the mouse aortic ring angiogenesis assay. Methods: The thoracic aorta of C57BL/6 mice were separated and all extraneous fat and connective tissues were removed. To remove the vascular adventitia, the aorta was digested with collagenase Ⅱ. The aorta were cut into rings～0.5 mm in width and the rings were embeded in the wells of a 96well plate coated with type Ⅰ collagen. The embedded rings were fed with 100 μL of DMEM culture medium supplemented with 10% FBS or 2.5% FBS and VEGF to attain a final concentration of 50 ng/mL. Inverted microscope was used to observe microvessel outgrowth of the aortic rings. The endothelial cells were assayed by BS1 lectinFITC immunofluorescence. Results: The number of microvessels of the aortic rings in the 96well plate was more than those in 24well plate（t=-6.000，P<0.05）. The number of microvessels of the aortic rings cultured with DMEM+10% fatal bovine serum was more than those cultured with DMEM+2.5% fatal bovine serum or DMEM+2.5% fatal bovine serum+VEGF. Microvessel outgrowth of the aortic rings started at 2 to 4 days and reached the peak at day 6 to 8 using the modified method. Conclusion: A great quantity, purity and high survival rate of the mouse aortic ring model of angiogenesis can be effectively obtained by coating plates with type Ⅰ collagen and enhancing the concentration of FBS.