马钱子对人肝癌细胞生长的影响及其作用机制

目的 探讨马钱子对人肝癌细胞生长影响及作用机制.方法 体外培养人肝癌细胞SMMC-7721,分别加入5％、10％、20％的马钱子药物血清,噻唑蓝(MTT)比色法测定对人肝癌细胞SMMC-7721的生长抑制作用.20％马钱子药物血清作用肝癌细胞SMMC-77210、24、48 h后,分别收集细胞,提取蛋白和总RNA,应用Westem blot和荧光定量聚合酶链反应(PCR)检测Fas、Cyclin D1、PCNA蛋白和mRNA表达.结果 20％马钱子药物血清对人肝癌细胞72 h的生长抑制率为(54.94±8.19)％,与5％、10％浓度抑制率(19.928±7.653)％、(29.020 ±6.100)％比较差异有统计学意义(F=18.23,P＜0.01).药物血清作用24、48 h后,肝癌细胞SMMC-7721 Fas蛋白表达分别为(0.302±0.009)、(0.399±0.021),与对照组(0.226±0.022)比较,差异有统计学意义(F=68.25,P＜0.05);药物血清作用48 h后,肝癌细胞SMMC-7721 PCNA蛋白表达为(0.7457±0.0386),与对照组、24 h(0.8529±0.0099、0.9016±0.0139)比较,差异有统计学意义(P＜0.05);药物血清作用24、48 h后,肝癌细胞SMMC-7721 Cyclin D1 mRNA表达分别为0.045 680 ±0.038 130、0.026 714±0.019934,与对照组(0.145246±0.048543)比较,差异有统计学意义(F=8.671,P＜0.05);药物血清作用24、48 h后,肝癌细胞SMMC-7721 Cyclin D1蛋白表达,PCNA、Fas mRNA表达变化差异无统计学意义(F =68.25,P＞0.05).结论 马钱子具有抑制人肝癌细胞增殖作用,其抑制效应与药物血清浓度成正相关;通过上调肝癌细胞Fas蛋白和下调PCNA蛋白及Cyclin D1 mRNA表达,是马钱子抑制肝癌细胞增殖的机制之一.     

马钱子碱纳米微粒抗肝细胞癌作用的实验研究

目的 观察马钱子碱纳米微粒的抗肝细胞癌作用.方法 采用超声乳化法研制羧基化聚乙二醇-聚乳酸嵌段共聚物马钱子碱纳米微粒,体外观察其对肝癌细胞SMMC-7721生长的影响;采用Western blotting和FQ-PCR检测药物作用72 h对肝癌细胞凋亡相关蛋白Fas蛋白和mRNA表达水平的影响.结果 马钱子碱纳米微粒平均粒径(146±96) nm,载药率:4.2％,包封率:67％.随药物浓度的增加,肝癌细胞生长抑制作用明显,呈现剂量依赖性.5-FU、马钱子碱和马钱子碱纳米微粒对肝癌细胞生长的IC50分别为16.7 μg/ml、90.3 μg/ml和164.9 μg/ml,三者之间差异显著(P＜0.05).与空白纳米微粒相比,马钱子碱纳米微粒显著诱导肝癌细胞Fas蛋白和mRNA表达,且320 μg/ml剂量诱导表达显著高于160 μg/ml (P＜0.05).结论 马钱子碱纳米微粒显著抑制肝癌细胞生长,诱导肝癌细胞凋亡,可能与上调肝癌细胞Fas蛋白和mRNA表达有关.马钱子碱纳米微粒是一种具有潜在发展前景的肝癌治疗药物.     

肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P＜0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在肝癌SP细胞中的含量分别为(92.65±3.92)%、(12.75±1.62)%、(17.35±2.31)%、(9.57±1.71)%和(28.39±5.28)%,在NSP细胞中的含量分别为(0.26±0.06)%、(2.51±0.17)%、(1.74±0.38)%、(1.52±0.41)%和(3.37±1.02)%,差异有统计学意义(P＜0.01).结论 肝癌SMMC-7721细胞中的SP细胞可能富集了肝癌干细胞,联合应用多种干细胞标记筛选肝癌SP细胞可能会获得纯化的肝癌干细胞.

Abstract：

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P ＜0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P ＜ 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.     

携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞凋亡的影响

目的 观察携带人mda-7/IL-24的溶瘤腺病毒SG600-IL24对各种不同转移潜能肝癌细胞HepG2、SMMC7721、MHCC97L和正常肝细胞LO2的作用.方法将携带人mda-7/IL-24的溶瘤腺病毒SG600-IL24分别感染人肝癌细胞系HepG2、SMMC7721、MHCC97L和正常肝细胞LO2,逆转录-聚合酶链式反应(RT-PCR)、酶联免疫吸附试验(ELISA)、Western blot检测mda-7/IL-24基因和蛋白的表达,MTT观察肝癌细胞的生长抑制,Hoechst33258和流式细胞仪(FCM)检测细胞的凋亡,流式细胞仪(FCM)检测细胞周期.结果 RT-PCR和Western blot显示mda-7/IL-24在肝癌细胞和正常肝细胞中高表达,ELISA提示细胞培养上清液中mda-7/IL-24蛋白也明显增加.MTT和流式细胞仪提示mda-7/IL-24能明显抑制肝癌细胞的生长(生长抑制率分别为75%±2.5%,86%±3.5%,72%±1.8%,HepG2∶F=5.86,SMMC7721∶F=7.98,MHCC97L∶F=5.13,均P<0.01),促进肝癌细胞的凋亡(凋亡抑制率分别为56.5%±4.0%,34.4%±2.0%,43.3%±2.5%,HepG2∶F=203.4,SMMC7721∶F=130.5,MHCC97L∶F=160.6,均P<0.01),阻滞肝癌细胞在G2/M期(G2/M期阻滞分别为35.4%±4.2%,40.5%±5.0%,42.0%±5.0%,HepG2∶F=112.5,SMMC7721∶F=133.2,MHCC97L∶F=145.5,均P<0.01),而对正常肝细胞没有明显的促进凋亡和增殖阻滞作用.结论 溶瘤腺病毒SG600-IL24能特异性杀伤不同转移潜能人肝癌细胞和诱导凋亡,促进肝癌细胞增殖阻滞,而对正常肝细胞无明显促进凋亡和增殖阻滞作用.

Abstract：

Objective To investigate the effect of oncolytic adenovirus vector SG600-IL24expressing human melanoma differentiation associated gene-7 (mda-7/IL-24) on hepatocellular carcinoma cell lines with different metastatic potential of HepG2, SMMC7721, MHCC97L and normal liver cell line LO2. Methods The oncolytic adenovirus SG600-IL24 which carrying mda-7/IL-24 gene was transfected into hepatocellular carcinoma cell lines and normal liver cell line. The mRNA and protein expression of mda7/IL-24 in HepG2, SMMC7721, MHCC97L and LO2 cell lines was confirmed by RT-PCR,ELISA assay and Western blot respectively. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst33258 and flow cytometry were studied to indicate the apoptosis effects. Results It was confirmed by RT-PCR, ELISA assay and Western-blot that the exogenous mda-7/IL-24 gene was highly expressed in HepG2, SMMC7721, MHCC97L and LO2 cell lines. MTT and apoptosis detection indicated that MDA-7/IL-24 can induce the growth suppression (the inhibition rate was 75% ±2. 5% ,86% ±3. 5% ,and promotes apoptosis ( the apoptosis rate was 56. 5% ± 4. 0% , 34. 4% ± 2. 0% , 43. 3% ± 2. 5%cell lines at G2/M phase ( the blocking rate was 35. 4% ± 4. 2% , 40. 5% ± 5. 0% , 42. 0% ± 5. 0%metastatic potential hepatocellular carcinoma cell lines but not in normal liver cell line.Conclusions Oncolytic adenovirus vector SG600-IL24 can selectively induce growth suppression, promote apoptosis in hepatocellular carcinoma lines in vitro but not in normal liver cell LO2.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

二氯乙酸盐对肺动脉高压大鼠肺组织KV1.5表达的影响

目的 探讨二氯乙酸盐对肺动脉高压大鼠肺组织Kv1.5表达的影响.方法 雄性SD大鼠32只,8周龄,体重200～250 g,采用随机数字表法,将大鼠随机分为4组(n=8):正常对照组(C组)、二氯乙酸盐对照组(D组)、肺动脉高压组(PAH组)和二氯乙酸盐治疗组(PD组).采用左肺切除术联合皮下注射野百合碱60mg/kg的方法制备肺动脉高压模型.PD组于皮下注射野百合碱后,给予二氯乙酸盐80mg/kg灌胃,1次/d,连续28 d,PAH组给予等量生理盐水;D组不制备模型,给予相同剂量二氯乙酸盐灌胃.于皮下注射野百合碱后28 d时测定肺动脉压(PAP),随后处死,取肺组织,计算肺小动脉中膜厚度百分比和右心室肥厚指数,采用Western blot法检测增殖细胞核抗原(PCNA)和Kv1.5蛋白的表达水平,采用RT-PCR法检测Kv1.5 mRNA的表达水平.结果 与C组比较,PAH组和PD组PAP、中膜厚度百分比及右心室肥厚指数升高,肺组织Kv1.5 mRNA及其蛋白表达下调,PCNA表达上调(P＜0.05),D组上述指标差异无统计学意义(P＞0.05);与PAH组比较,PD组PAP、中膜厚度百分比及右心室肥厚指数降低,肺组织Kv1.5mRNA及其蛋白表达上调,PCNA表达下调(P＜0.05).结论 二氯乙酸盐减轻大鼠肺动脉高压与上调肺组织Kv1.5表达,抑制肺血管重构有关.

Abstract：

Objective To investigate the effect of dichloroacetate on the expression of Kv1.5 in a rat model of pulmonary arterial hypertension (PAH) .Methods Thirty-two male SD rats weighing 200-250 g were randomly divided into 4 groups ( n = 8 each): normal control group (group C), dichloroacetate control group (group D),PAH group, and PAH + dichloroacetate group (group PD). PAH was induced by left lung resection combined with subcutaneous injection of monocrotaline 60 mg/kg in PAH and PD groups. In group PD, dichloroacetate 80 mg/kg was given through a gastric tube into stomach once a day for 28 consecutive days after monocrotaline injection,while the equal volume of normal saline was given instead of dichloroacetate in group PAH. Group D only received dichloroacetate 80 mg/kg through a gastric tube into stomach once a day for 28 consecutive days. Pulmonary arterial pressure (PAP) was measured at day 28 after monocrotaline injection. The rats were then sacrificed and lung tissues were removed to calculate the percentage of thickness of the tunica media of pulmonary artery and right venicular hypertrophy index and to determine the proliferating cell nuclear antigen (PCNA) and Kv1.5 protein expression (by Western blot) and Kv1.5 mRNA expression (by RT-PCR).Results Compared with group C, the PAP,percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly increased, Kv1.5 mRNA and protein expression was down-regulated and PCNA expression was up-regulated in groups PAH and PD ( P ＜ 0.05). Compared with group PAH, the PAP, percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly decreased, Kv1.5 mRNA and protein expression was up-regulated and PCNA expression was down-regulated in group PD (P ＜ 0.05). There was no significant difference in the indexes mentioned above between group C and group D ( P ＞ 0.05). Conclusion Dichloroacetat alleviates PAH through upregulating Kv1.5 expression in lung tissues and inhibiting pulmonary vascular remodeling in rats.     

表达IL-24基因的溶瘤腺病毒对肝癌细胞的抑制作用

目的 研究携带IL-24基因的溶瘤腺病毒对肝癌细胞抑制生长和转移的作用.方法 构建Ad.HS4.AFP.E1A/IL-24,病毒的复制由人甲胎蛋白启动子控制,携带IL-24基因.检测病毒在不同细胞系中的选择性复制,以及对高转移潜能人肝癌细胞系MHCC97-H的生长抑制、诱导凋亡和抑制转移能力(侵袭、运动、黏附)的作用.结果 Ad.HS4.AFP.E1A/IL-24在肝癌细胞SMMC-7721、Hep3B和MHCC97-H中选择性表达,而不影响正常肝细胞L02(P＜0.05).Ad.HS4.AFP.E1A/IL-24可明显抑制MHCC97-H细胞的增殖,诱导其凋亡,并抑制其体外运动、侵袭和黏附能力(P＜0.01).RT-PCR和明胶酶谱显示其抑制肝癌作用与抑制MMP-2的表达有关.结论 携带IL-24基因的溶瘤腺病毒能够选择性抑制肝癌细胞的增殖并抑制其转移.

Abstract：

Objective To investigate the selective oncolytic role and antitumor action of a novel recombinant adenovirus containing E1A and IL-24 on hepatocellular carcinoma cell(HCC). Methods The recombinant adenovirus expressing IL-24 (Ad. HS4. AFP. E1A/IL-24) was constructed by using modified human alpha-fetoprotein (HS4-AFP) promoter to drive adenovirus E1A gene and II-24 gene.Cell Counting Kit-8 were performed to test the selective cytotoxicity of the virus in hepatocellular carcinoma cell lines SMMC-7721, Hep3B, MHCC97-H and hepatocyte cell line L02 . The mRNA and protein expression of IL-24 gene were detected by RT-PCR and western blot. Cell growth curves and Annexin V/PI assay were used to study cell proliferation and apoptosis of MHCC97-H. The anti-metastatic effects of the recombinant adenovirus were evaluated in cell adhesion, migration, and cell motion. Matrix metalloproteinase-2 (MMP-2) expression was examined by RT-PCR and zymography.Results Selective replications of Ad. HS4. AFP. E1A/IL-24 adenovirus were observed in over expression AFP cell line MHCC97-H, a highly metastatic potential HCC cell line but not in hepatocyte cell line L02. The mRNA and protein of IL-24 were also over expressed in MHCC97-H. This recombinant adenovirus also showed the significant oncolytic action on MHCC97-H but not on L02 (P＜0. 05). Besides, the recombinant adenovirus significantly inhibited MHCC97-H metastatic potential such as cell adhesion, migration and invasion as well(P＜0.01). Conclusion The selective oncolytic adenovirus expressing E1A and II-24 has a selective antitumor effect and play an inhibitory role in metastasis of HCC.     

真核始动因子4E反义寡核苷酸对人膀胱癌BIU-87细胞中eIF4E和肝素酶表达的影响

目的 探讨真核始动因子4E(eIF4E)反义寡核苷酸(ASODN)对人膀胱癌BIU-87细胞株中eIF4E及肝素酶(HPA)蛋白、mRNA表达的影响. 方法 采用脂质体介导方法分别将2.5、5.0及7.5 μg/ml eIF4E ASODN转染膀胱癌BIU-87细胞,并设立细胞对照组(不转染)、空白对照组(转染空脂质体)及无关对照组(转染无关对照ASODN),分别转染24、48及72 h,采用原位杂交、免疫细胞化学方法分别检测各组BIU-87细胞中eIF4E及HPA蛋白、mRNA表达. 结果 eIF4E ASODN各转染组细胞中eIF4E蛋白及mRNA表达量均较对照组降低(蛋白:2.5 μg/ml组分别为3.55±0.52、3.52 ±0.51、3.22 ±0.44;5.0 μg/ml组分别为3.43±0.47、3.41±0.46、3.19±0.41;7.5 μg/ml组分别为2.36±0.39、2.33±0.37、2.05±0.30.mRNA:2.5 μg/ml组分别为3.19±0.41、3.13±0.39、2.90±0.38;5.0μg/ml组分别为3.07±0.39、2.94 ±0.38、2.27±0.37;7.5 μg/ml组分别为2.22±0.37、2.06±0.30、1.95 ±0.29),与细胞对照组、空白对照组和无关对照组相应时间段、相应浓度组两两比较差异均有统计学意义(P＜0.05),且具有时间和浓度依赖性.eIF4E ASODN各转染组细胞中HPA蛋白及mRNA表达量均较对照组降低(蛋白:2.5 μg/ml组分别为4.44±0.55、4.40±0.54、3.99±0.52;5.0μ g/ml组分别为4.41 ±0.55、4.21±0.53、3.77±0.50;7.5 μg/ml组分别为4.02±0.52、3.98±0.52、2.32±0.37.mRNA:2.5μg/ml组分别为4.12±0.51、3.75±0.50、3.63±0.45;5.0 μg/ml组分别为4.00 ±0.51、3.71±0.50、3.54 ±0.44;7.5 μg/ml组分别为3.87±0.52、3.61 ±0.49、2.08 ±0.30),与细胞对照组、空白对照组和无关对照组两两比较差异均有统计学意义(P＜0.05),且具有时间和浓度依赖性.eIF4E ASODN对HPA蛋白、mRNA表达的抑制效应和对eIF4E蛋白、mRNA表达的抑制效应呈正相关关系(HPA蛋白r=9.23,mRNA r=9.59,P值均＜0.01). 结论eIF4E ASODN可下调膀胱癌BIU-87细胞中eIF4E、HPA蛋白及mRNA的表达,抑制肿瘤细胞的生长、浸润及转移.     

脂多糖对小鼠肺成纤维细胞活化的影响

目的 评价脂多糖(LPS)对小鼠肺成纤维细胞活化的影响.方法 原代培养的小鼠肺成纤维细胞,接种于96孔培养板,采用随机数字表法,将其随机分为2组,正常对照组(C组,外=6)不作任何处理,LPS组(n=24)加入LPS 1μg/ml,分别于LPS孵育3、6、24、72 h时取6孔(C组于培养72 h时),收集细胞,采用实时PCR法测定Ⅰ型前胶原mRNA、α-平滑肌肌动蛋白(α-SMA)mRNA、Toll样受体4(TLR4)mRNA和整合素β1 mRNA的表达水平.结果 与C组比较,LPS组LPS孵育3、6和24 h时Ⅰ型前胶原mRNA、α-SMA mRNA、TLR4 mRNA和整合素β1 mRNA的表达水平差异无统计学意义(P＞0.05),LPS孵育72 h时上述指标表达水平上调(P＜0.05).结论 在急性肺损伤的早期LPS一方面可直接活化小鼠肺成纤维细胞,导致肺纤维化;另一方面可上调TLR4和整合素β1的表达,增加细胞对LPS的反应性,从而加速小鼠肺成纤维细胞的活化,促进肺纤维化.

Abstract：

Objective To investigate the effect of lipopolysaccharide (LPS) on the activation of mouse lung fibroblasts. Methods Primary cultured mouse lung fibroblasts were incubated in 96 well plates and randomly divided into 2 groups: control group ( group C, n = 6) and LPS group ( n = 24). The fibroblasts were cultured for 72 h in group C. LPS 1 μg/ml was added and then the fibroblasts were incubated for 72 h in group LPS. The expression of type Ⅰ procollagen mRNA, α-smooth muscle actin (α-SMA) mRNA, Toll-like receptor 4 (TLR4)mRNA and integrin β1 mRNA was determined using real-time PCR at 3, 6, 24 and 72 h of incubation (6 wells at each time point). Results Compared with group C, there was no significant change in the expression of type Ⅰ procollagen mRNA, α-SMA mRNA, TLR4 mRNA and integrin β1 mRNA at 3, 6 and 24 h of incubation ( P ＞0.05), but the parameters mentioned above were significantly up-regulated at 72 h of incubation in group LPS ( P ＜ 0.05). Conclusion In the early acute lung injury, LPS leads to pulmonary fibrosis through activating lung fibroblasts directly, and also accelerates the activation of lung fibroblasts and promotes the process of pulmonary fibrosis through up-regulating the expression of TLR4 and integrin β1 in mice.     

rhALR在大鼠部分肝移植术后肝再生中的作用及其机制研究

目的 探讨大鼠部分肝移植术后腹腔注射重组人肝再生增强因子(recombinant human augmenter ofliver regeneration,rhALR)促进肝移植术后肝细胞再生的作用及其机制.方法 建立大鼠原位60% 肝脏移植模型,部分肝移植术后腹腔注射rhALR 40 μg/kg 2 次/d(实验组,对照组注射等体积的注射用水);取术后第1、2、3、5、7 天大鼠血清及肝组织,检测血清谷丙转氨酶(ALT),测定肝细胞PCNA LI 的变化,RT-PCR 检测肝组织IL-6 mRNA Cyclin-D1 mRNA 的表达.结果 实验组(A 组)与对照组(B 组)比较,术后第1、2 天A 组血清ALT 显著低于B 组.A 组术后第1、2、3、5 天的PCNA LI 均明显高于B 组同一时点的PCNA LI(P<0.05).A 组术后第2 天的肝细胞AI 明显低于B 组同一时点的肝细胞AI(P<0.05);A 组术后肝组织IL-6 mRNA 的表达与B 组在各观察时点间比较均无统计学差异(P>0.05);A 组术后第1 天肝组织Cyclin D1 mRNA 的表达明显高于B 组同时点的表达(P<0.05),其余观察时点无明显差异.结论 大鼠部分肝移植术后应用rhALR 可以明显促进肝细胞的再生,降低血清ALT,稳定肝细胞膜,保护肝细胞功能.rhALR 促进大鼠部分肝移植术后的肝再生可能并不是通过上调启动阶段重要因子IL-6 mRNA 的表达发挥作用,而是通过上调增殖阶段重要因子CyclinD1mRNA 的表达发挥作用.     

MIF和Cyclin D1在肝细胞癌中的表达

目的 探讨巨噬细胞游走抑制因子(macrophage migration inhibitory factor,MIF)和细胞周期蛋白D1(cyclin D1)在原发性肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及两者在肝癌发病机制及细胞周期调控中的关系.方法 应用定量PCR及Westem blot技术检测MIF和Cyclin D1在肝癌组织和癌旁组织的表达.化学合成MIF siRNA,将PLC细胞和HepG2细胞分成对照组、MIF siRNA 50 nmol/L干预组、MIF siRNA 1130 nmol/L干预组,脂质体法转染肝癌细胞PLC和HepG2,定量PCR技术和Western blot检测MIF siRNA干扰后MIF和Cyclin D1 mRNA和蛋白的表达变化.结果 MIF和Cyclin D1蛋白在肝癌组织中的相对表达量为0.825±0.13和0.843±0.104;MIF和Cyclin D1 mRNA在肝癌组织中的相对表达量为癌旁组织的(7.31±1.85)倍和(4.27±1.05)倍,与癌旁组织相比,差异均具有统计学意义(FMIF=15.5,P<0.01;fCyclin D1=87.5,P<0.01).应用小RNA干扰技术转染PLC和HepG2肝癌细胞后MIF mRNA分别下调71.2%±7.2%、87.4%±2.9%、74.3%±8.9%、88.4%±4.6%(FPLC=315.5,P<0.01;FHHepG2=201.2,P<0.01);MIF蛋白分别下调为0.33±0.03、0.11±0.02、0.81±0.08、0.36±0.02,并呈剂量依赖关系(FPLC=43.9,P<0.01;FHepG2:133.4,P<0.01).伴随MIF mRNA和蛋白的表达F调Cyclin D1 mRNA分别下调68.2%±3%、78.1%±1.4%、65.8%±4.7%、77.3%±2.6%(FPLC=1569,P<0.01;FHepG2=480.4,P<0.01);Cyclin D1蛋白下调0.28±0.06、0.15±0.03、0.44±0.04、0.13±0.02,亦呈剂量依赖关系,与对照组相比差异有统计学意义(P<0.01),两干预组相比差异有统计学意义(FPLC=35.5,P<0.01;FHepG2=114.7,P<0.01).结论 MIF和Cyclin D1在肝细胞癌中过表达,MIF可能在转录水平调控Cyclin D1的表达,并促使肿瘤细胞通过细胞周期检测点,继续增值和分化,导致肿瘤的形成.     

氧化低密度脂蛋白对大鼠肾脏系膜细胞增殖及LOX-1、TNF-α表达的影响

目的：应用MTT酶标分析法,检测在不同浓度、不同时长氧化低密度脂蛋白（ ox-LDL）干预下的大鼠肾小球系膜细胞（ RMC）的增殖情况以及不同浓度ox-LDL干预下LOX-1和TNF-α的表达,探讨其相互关系。方法：体外培养RMC,使用不同浓度ox-LDL在不同时间对RMC干预,经MTT法检测,并进行分组分析。用浓度为5μg/ml和50μg/ml的ox-LDL干预RMC 24 h,使用RT-PCR法和Western blot法检测LOX-1和TNF-α的mRNA和蛋白表达量,并进行分析比较。结果：Ox-LDL干预RMC 在时间点为24 h的结果显示,在1-30μg/ml范围内呈生长促进效应,浓度大于30μg/ml后呈生长抑制效应;干预48 h组对细胞效应趋势基本与作用24 h后一致,但生长抑制效应从30μg/ml开始;而72 h组,ox-LDL在各个浓度均显示出生长抑制效应。5μg/ml及50μg/ml浓度ox-LDL干预RMC后,均可使LOX-1和TNF-α的mRNA和蛋白表达升高,且50μg/ml较5μg/ml效果明显,结果差异具有统计学意义（P〈0.05）。结论：Ox-LDL对RMC增殖影响具有浓度及时间依赖性。较低及较高浓度ox-LDL均能导致RMC中LOX-1和TNF-α的表达增高,且浓度较高时效果更明显。其机制可能为ox-LDL导致LOX-1表达增多,引起炎症因子的产生和趋化,发生细胞损伤,提示脂质代谢紊乱在肾脏疾病中的重要作用。     

细胞周期素D1反义cDNA治疗肝癌的研究

目的 通过基因反义封闭技术抑制细胞周期素D1（CydinD1）的表达，研究其对肝癌细胞增殖以及成瘤性的影响。方法 以肝癌HepG2细胞株为研究对象，通过转染可表达CyclinD1反义互补脱氧核苷酸（AScDNA）的质粒后，观察CyclinD1反义cDNA对肝癌细胞CyclinD1基因表达、体外增殖活性及裸鼠体内成瘤性的影响。结果 噻唑蓝（MTT）法检测细胞增殖活性显示转染表达反义CydinD1的质粒后，HepG2细胞的增殖受到抑制．抑制作用在48h左右最强；逆转录-聚合酶链反应（RT-PCR）检测显示CyclinD1 mRNA基因的表达明显被抑制；间接免疫荧光检测结果显示CyclinD1蛋白表达显著降低；流式细胞仪检测结果显示G0／G1期的细胞比例增高，G2＋M和S期的细胞比例下降，HepG2细胞周期在G1期被阻滞；裸鼠成瘤试验显示肝癌HepG2细胞的成瘤性受到明显抑制。结论 CyclinD1反义cDNA可以特异性的抑制肝癌HepG2细胞株CyclinD1蛋白的表达，从而调控细胞周期，抑制肝癌细胞增殖及体内成瘤性。CyclinD1反义cDNA对于肝细胞癌的生物治疗具有一定的应用前景。     

Hint1基因抑制HepG2细胞AP-1转录因子活性的实验研究

目的 探索体外过表达Hint1基因对人肝肿瘤HepG2细胞AP-1转录因子活性的影响.方法 分子克隆获得Hint1基因序列,构建pHA-Hint1重组融合基因表达载体.阳离子脂质体介导,pHA-Hint1转导人肝母细胞瘤HepG2细胞,RT-PCR和Western blot检测HepG2细胞中外源HA-Hint1表达.pHA-Hint1质粒、启动子荧光素酶报告质粒AP-1/Luc及巨细胞病毒-β-半乳糖酶报告质粒(β-gel)共转导HepG2细胞.36 h后测定荧光素酶活性及β-gel活性.结果 重组质粒经EcoR Ⅰ和BamH Ⅰ双酶切,获约100 bp特异条带,测序证实成功构建pHA-Hint1表达载体.pHA-Hint1及空载体pcDNA3/HA转导HepG2细胞,半定量RT-PCR结果显示pHA-Hint1组Hint1mRNA表达高于空载体组(t=3.89,P<0.05);Western blot结果显示pHA-Hint1组HA标签蛋白表达高于空载体组(t=3.12,P<0.05).AP-1转录因子活性检测结果:pHA-Hint1终浓度为0 μg/ml,0.5 μg/ml,1.0 μg/ml,1.5 μg/ml,2.0 μg/ml时,AP-1转录因子相对活性(106)为:5.12±0025、4.24±0.74、3.43±0.31、2.62±0.48、2.09±0.21.随着pHA-Hint1浓度增加,荧光素酶活性呈明显下降,当pHA-Hint1浓度达到1.5 μg及2.0 μg时,差异有统计学意义(F=72.009,P<0.05).结论 过表达Hint1基因可抑制HepG2细胞AP-1转录因子活性.     

异丙酚预先给药对内毒素诱导大鼠肾小球血管内皮细胞通透性升高的影响

目的 探讨异丙酚预先给药对脂多糖(LPS)诱导大鼠肾小球血管内皮细胞通透性升高的影响.方法 分离、培养SD大鼠肾小球血管内皮细胞,以1×106/ml的密度接种于24孔培养板(200 μl/孔)和transwell小室(100 μl/室),采用随机数字表法,将其随机分为6组(n=10),正常对照组(C组)不作任何处理;脂肪乳对照组(I 组)加入10%脂肪乳4 μg/ml;异丙酚组(P组)加入异丙酚4μg/ml;LPS组(L组):加入LPS 10μg/ml;LPS+脂肪乳组(L+I组)加入10%脂肪乳4 μg/ml及LPS 10μg/ml;LPS+异丙酚组(L+P组)加入异丙酚4μg/ml 及LPS 10 μg/ml.于加入LPS前30 min加入脂肪乳或异丙酚,药物的浓度均为终浓度.加入LPS后6 h,收集细胞,采用逆转录-聚合酶链反应测定血管内皮细胞生长因子(VEGF)mRNA表达水平;收集上清液,采用酶联免疫吸附法测定VEGF浓度;测定血管内皮细胞通透性.结果 与C组比较,L组、L+I组和L+P组VEGF mRNA表达上调,上清液中VEGF浓度和血管内皮细胞通透性增加(P＜0.05),而I组和P组上述指标差异无统计学意义(P＞O.05).与L组比较,L+P组VEGF mRNA表达下凋,上清液中VEGF浓度和血管内皮细胞通透性降低(P＜0.05),L+I组上述指标差异无统计学意义(P＞0.05).上清液中VEGF浓度与血管内皮细胞通透性呈正相关(r=0.833,P＜0.05).结论 异丙酚预先给药可抑制LPS诱导大鼠肾小球血管内皮细胞通透性升高,其机制与下调VEGF表达有关.

Abstract：

Objective To investigate the influence of propofol pretreatment on the increased glomerular endothelial cell permeability induced by lipopolysaccharide (LPS) in rats.Methods Glomerular endothelial cells isolated from SD rats were cultured in 24-well plates(200 μl/well) and transwell filters (100 μl/filter) at 1×106/ml and assigned into 6 groups (n=10 each):control group (group C) , introlipid group (group I), propofol group (group P) , LPS group (group L), LPS+introlipid group (group L+I) and LPS+propofol group (group L +P). In group I, 10% introlipid 4 μg/ml was added. In group P, 4 μg/ml propofol was added. In group L, 10 μg/ml LPS was added. In group L+I, 10% introlipid 4 fig/ml combined with 10 μg/ml LPS was added. In group L+ P, 4 μg/ml propofol combined with LPS 10 μg/ml was added. Introlipid or propofol was added 30 min before the administration of LPS and the corresponding concentrations mentioned above were all final concentrations.After 6 h incubation with LPS, the cells were collected for measurement of vascular endothelial growth factor (VEGF) mRNA expression using RT-PCR. The supernatant was collected for determination of the VEGF concentration by ELJSA. The endothelial cell permeability was determined. Results Compared with group C, the expression of VEGF mRNA was up-regulated and the VEGF concentration and endothelial cell permeability were significantly increased in L, L+I and L + P groups (P＜0.05 ) ,but no significant change was found in the parameters mentioned above in I and P groups (P＞0.05). Compared with group L, the expression of VEGF mRNA was downregulated and the VEGF concentration and endothelial cell permeability were significantly decreased in L+P group (P＜0.05), but no significant change was found in the parameters mentioned above in group L+I(P＞0.05). A positive correlation existed between the concentration of VEGF and the permeability of endothelial cells(r= 0.833,P＜0.05).Conclusion Propofol pretreatment can decrease the increased glomerular endothelial cell permeability induced by LPS probably through down-regulation of VEGF expression.