高表达蛋白hSav1对人胚肾HEK293增殖能力的影响

Objective To elucidate the effects of human Salvador 1 (hSav1 ) on cell proliferation of human embryonic kidney cell line HEK293. Methods The plasmid CFP-N1-hSav1 was constructed and transfected into HEK293 cells with lipofectamine 2000. The transfection efficiency was detected by fluorescent microscopy. The effects of hSav1 on cell proliferation were measured by MTT and BrdU incorporation. Results The transfection efficiency was about 70% -80%. The MTT results showed that the inhibition rate of cell proliferation in transfected group at the 12th, 24th, 36th, and 48th h was 2% , 5% , 15% , and 23% , respectively;while that in the control group was 2% , 3% , 2% , and 2% respectively. The BrdU incorporation revealed that the BrdU incorporation rate in transfected group (12. 9 ±5. 3)% was significantly lower than in control group (27.3±3.8)% (P<0.05). Conclusion hSav1 is a newly identified protein that can cause cell proliferation inhibition in HEK293 cells.     

高表达蛋白hSav1对人胚肾HEK293增殖能力的影响

Objective To elucidate the effects of human Salvador 1 (hSav1 ) on cell proliferation of human embryonic kidney cell line HEK293. Methods The plasmid CFP-N1-hSav1 was constructed and transfected into HEK293 cells with lipofectamine 2000. The transfection efficiency was detected by fluorescent microscopy. The effects of hSav1 on cell proliferation were measured by MTT and BrdU incorporation. Results The transfection efficiency was about 70% -80%. The MTT results showed that the inhibition rate of cell proliferation in transfected group at the 12th, 24th, 36th, and 48th h was 2% , 5% , 15% , and 23% , respectively;while that in the control group was 2% , 3% , 2% , and 2% respectively. The BrdU incorporation revealed that the BrdU incorporation rate in transfected group (12. 9 ±5. 3)% was significantly lower than in control group (27.3±3.8)% (P<0.05). Conclusion hSav1 is a newly identified protein that can cause cell proliferation inhibition in HEK293 cells.     

缓激肽促进人肺腺癌细胞株A549的侵袭及其机制

目的 观察缓激肽(BK)对人肺癌细胞株A549迁移、侵袭能力的影响.方法 应用划痕愈合实验和Transwell实验检测BK对40例人肺癌细胞株A549的划痕愈合和运动侵袭能力.通过Western blot分析刺激和抑制缓激肽各20例后基质金属蛋白酶(MMP)-2、MMP-9和E-钙黏素(E-Cadherin)的变化并研究其机制.结果 划痕实验后分别测量愈合距离,结果 示BK组24h和48h后迁移细胞数分别为69.2±3.3、94.1±2.9,而阻断其受体或者ERK1/2通路则迁移细胞数分别为51.2±2.1、73.2±2.7和47.5±3.4、77.6±3.8.Transwell实验检测BK对A549侵袭能力的影响结果 显示BK可以明显促进A549的侵袭能力.阻断BK受体或ERK1/2通路则可以抑制其侵袭能力.结论 缓激肽促进人肺腺癌细胞株A549的迁移和侵袭能力.

Abstract：

Objective To investigate the effect of bradykinin (BK) on human lung cancer cell line A549 migration,invasion ability. Methods Scratch Healing and Transwell assay of bradykinin were used to detect scratch healing and movement on A549 human lung cancer cells. Analysis the changes of matrix metalloproteinase (MMP)-2, MMP-9 and E-Cadherin after stimulate and suppress 20 cases respectively in the aim to describe the mechanism by Western blotting. Results In the former, BK group promote cell migration significantly,migrated cell was 69.2±3.3, 94.1±2.9 respectively after 24 h and 48 h,blocking its receptor or ERK1/2 pathway inhibit migration,migrated cell was 51.2±2.1, 73.2±2.7 and 47.5±3.4,77.6±3.8 after 24 h and 48 h. In the latter,Transwell assay of A549 BK showed invasion of A549 BK can significantly promote the invasion ability of A549 to penetrate the basement membrane of the cell. And blocking BK receptor or ERK1/2 pathway can inhibit the invasion. Conclusion Bradykinin promote human lung adenocarcinoma cell line A549 migration and invasion.     

不同浓度七氟醚对人肺腺癌A549细胞Survivin蛋白表达的影响

Objective To investigate the effects of different concentrations of sevoflurane on Survivin expression in human adenocarcinoma cell line A549. Methods A549 cells were obtained from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences and inoculated in 96 well culture plate. After being cultured for 24 h, the cells were randomly divided into 4 groups: Ⅰ , Ⅱ , Ⅲ and Ⅳ groups exposed to 95 % O2 -5 %CO2,1.7%, 3.4% and 5.1% sevoflurane respectively. A549 cells were exposed to sevoflurane for 2, 4 and 6 h respectively and then cultured for another 48 h in Ⅱ , Ⅲ and Ⅳ groups. Proliferation of A549 cells were measured by methyl thiazolyl tetrazolium (MTT) assay, and apoptosis was detected with flow cytometer at 48 h after 2, 4 and 6 h sevoflurane exposure. The expression of Survivin in A549 cells was determined by Western blot analysis at 48h after 4 h sevoflurane exposure. Results The rate of proliferation inhibition and percentage of apoptotic cells were significantly higher while the expression of Survivin was significantly lower in a concentration-dependent manner in Ⅱ , Ⅲ and Ⅳ groups as compared with group Ⅰ . Conclusion Sevoflurane can inhibit proliferation and induce apoptosis of A549 cells by inhibition of Survivin expression.     

不同浓度七氟醚对人肺腺癌A549细胞Survivin蛋白表达的影响

Objective To investigate the effects of different concentrations of sevoflurane on Survivin expression in human adenocarcinoma cell line A549. Methods A549 cells were obtained from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences and inoculated in 96 well culture plate. After being cultured for 24 h, the cells were randomly divided into 4 groups: Ⅰ , Ⅱ , Ⅲ and Ⅳ groups exposed to 95 % O2 -5 %CO2,1.7%, 3.4% and 5.1% sevoflurane respectively. A549 cells were exposed to sevoflurane for 2, 4 and 6 h respectively and then cultured for another 48 h in Ⅱ , Ⅲ and Ⅳ groups. Proliferation of A549 cells were measured by methyl thiazolyl tetrazolium (MTT) assay, and apoptosis was detected with flow cytometer at 48 h after 2, 4 and 6 h sevoflurane exposure. The expression of Survivin in A549 cells was determined by Western blot analysis at 48h after 4 h sevoflurane exposure. Results The rate of proliferation inhibition and percentage of apoptotic cells were significantly higher while the expression of Survivin was significantly lower in a concentration-dependent manner in Ⅱ , Ⅲ and Ⅳ groups as compared with group Ⅰ . Conclusion Sevoflurane can inhibit proliferation and induce apoptosis of A549 cells by inhibition of Survivin expression.     

不同浓度七氟醚对人肺腺癌A549细胞Survivin蛋白表达的影响

目的 评价不同浓度七氟醚对人肺腺癌A549细胞Survivin蛋白表达的影响.方法 人肺腺癌A549细胞接种于培养板培养24 h,采用随机数字表法,将人肺腺癌A549细胞随机分为4组:对照组(C组)、1.7%七氟醚组(S1组)、3.4%七氟醚组(S2组)和5.1%七氟醚组(S3组),每组18孔.S1-3组人肺腺癌A549细胞分别经1.7%、3.4%、5.1%七氟醚孵育2、4、6 h,C组通入95%O2-5%CO2混合气体2 L/min,每个时点随机取6孔,继续培养48 h,采用MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,Western blot法检测经七氟醚孵育4 h的人肺腺癌A549细胞Survivin蛋白表达水平.结果 C组、S1～3组入肺腺癌A549细胞增殖抑制率和凋亡率依次升高,Survivin蛋白表达依次下调(P<0.05).结论 七氟醚可通过下调Survivin蛋白表达抑制人肺腺癌A549细胞增殖和促进凋亡,其效应呈浓度依赖性.

Abstract：

Objective To investigate the effects of different concentrations of sevoflurane on Survivin expression in human adenocarcinoma cell line A549. Methods A549 cells were obtained from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences and inoculated in 96 well culture plate. After being cultured for 24 h, the cells were randomly divided into 4 groups: Ⅰ , Ⅱ , Ⅲ and Ⅳ groups exposed to 95 % O2 -5 %CO2,1.7%, 3.4% and 5.1% sevoflurane respectively. A549 cells were exposed to sevoflurane for 2, 4 and 6 h respectively and then cultured for another 48 h in Ⅱ , Ⅲ and Ⅳ groups. Proliferation of A549 cells were measured by methyl thiazolyl tetrazolium (MTT) assay, and apoptosis was detected with flow cytometer at 48 h after 2, 4 and 6 h sevoflurane exposure. The expression of Survivin in A549 cells was determined by Western blot analysis at 48h after 4 h sevoflurane exposure. Results The rate of proliferation inhibition and percentage of apoptotic cells were significantly higher while the expression of Survivin was significantly lower in a concentration-dependent manner in Ⅱ , Ⅲ and Ⅳ groups as compared with group Ⅰ . Conclusion Sevoflurane can inhibit proliferation and induce apoptosis of A549 cells by inhibition of Survivin expression.     

CPB诱发犬急性肺损伤时肺组织转化生长因子β1 mRNA表达的变化

Objective To observe the changes in the transforming growth factor-beta 1 (TGF-β1 ) mRNA expression in the lung in a dog model of cardiopulmonary bypass(CPB)-induced acute lung injury. Methods Thirty-six healthy adult mongrel dogs of both sexes weighing 15-16 kg were randomly assigned into control group and CPB group ( n = 18 each) . Lung injury was produced by CPB according to the method described by Williams. Six animals were killed at each of the following time points: before CPB (T0 ) and 30 and 60 min after termination of CPB (T1 , T2) in each group. Lung specimens were obtained for microscopic examination and determination of TGF-β1 mRNA expression (by RT-PCR) and MDA content. The lungs were lavaged and the protein concentration in the brancho-alveolar lavage fluid (BALF) was determined and pulmonary permeability index (PPI) was calculated. Results Microscopic examination showed massive inflammatory cell infiltration, alveolar capillary dilatation, congestion, widened alveolar septum, massive RBC in the alveolar space and focal atelectasis in the lung in CPB group. The TGF-β1 mRNA expression and MDA content and PPI were significantly higher in CPB group than in control group. The TGF-β1 mRNA expression and MDA was positively correlated to PPI (MDA: r = 0.867, P < 0.01; PPI: r = 0.821, P < 0.01) . Conclusion TGF-β1 mRNA expression in the lung is significantly up-regulated after CPB and is an important factor contributing to CPB-induced acute lung injury.     

CPB诱发犬急性肺损伤时肺组织转化生长因子β1 mRNA表达的变化

Objective To observe the changes in the transforming growth factor-beta 1 (TGF-β1 ) mRNA expression in the lung in a dog model of cardiopulmonary bypass(CPB)-induced acute lung injury. Methods Thirty-six healthy adult mongrel dogs of both sexes weighing 15-16 kg were randomly assigned into control group and CPB group ( n = 18 each) . Lung injury was produced by CPB according to the method described by Williams. Six animals were killed at each of the following time points: before CPB (T0 ) and 30 and 60 min after termination of CPB (T1 , T2) in each group. Lung specimens were obtained for microscopic examination and determination of TGF-β1 mRNA expression (by RT-PCR) and MDA content. The lungs were lavaged and the protein concentration in the brancho-alveolar lavage fluid (BALF) was determined and pulmonary permeability index (PPI) was calculated. Results Microscopic examination showed massive inflammatory cell infiltration, alveolar capillary dilatation, congestion, widened alveolar septum, massive RBC in the alveolar space and focal atelectasis in the lung in CPB group. The TGF-β1 mRNA expression and MDA content and PPI were significantly higher in CPB group than in control group. The TGF-β1 mRNA expression and MDA was positively correlated to PPI (MDA: r = 0.867, P < 0.01; PPI: r = 0.821, P < 0.01) . Conclusion TGF-β1 mRNA expression in the lung is significantly up-regulated after CPB and is an important factor contributing to CPB-induced acute lung injury.     

5-氟尿嘧啶对瘢痕疙瘩成纤维细胞增殖与凋亡机制影响的初步探讨

Objective To investigate the of 5-fluorouracil effects on the expression of Smad7,TGF-β receptorⅠ,Bcl-2 and Bax in keloid fibroblasts.Methods After primary culture of keloid fibroblasts,4-6 passages of cells were inoculated in 5 different concentrations of 5-fluorouracil(10,20,40,80,160μmol/L)for 24,48 and 72 hours.Proliferative ability of keloid fibroblasts was detected by MTT assay.Expression of Smad 7,TGF-βreceptorⅠ,Bcl-2 and Bax in keloid fibroblasts was measured by Western blot.Results During MTT,5-fluorouracil did not affect cell viability at 24 hour at the concentration of 10 and 20 μmol/L.Compared with the control group,no significant difference was detected(P＞0.05).At other concentrations,fibroblast death was visible in each group(P＜0.01).Western blot analysis showed that the expression of Smad7 significantly decreased and the expression of TGF-β receptor Ⅰ significantly increased in the TGF-β1 group compared with the blank control group(P＜0.0 1).5-fluorouracil could significantly enhance the expression of Smad7(P＜0.01).There was a remarkable decrease of the Bcl-2 expression and marked increase of the Bax expression in different concentrations of 5-fluorouracil compared with the control group(P＜0.05).But,5-fluorouracil did not show any effect on the synthesis of TGF-β receptor Ⅰ.Conclusion 5-fluorouracil could inhibit proliferation and induce apoptosis on human keloid fibroblasts in vitro.     

缺氧诱导因子-1α反义寡核苷酸体外对胶质瘤U87细胞增殖和侵袭的影响

目的 观察缺氧诱导因子-1α(HIF-1α)反义寡核苷酸体外对人胶质瘤U87细胞增殖、凋亡和侵袭的影响.方法 人工合成的HIF-1α反义短发夹经阳离子脂质体包裹后瞬时转染人胶质瘤细胞株U87,采用Western blot法检测转染后胶质瘤细胞HIF-1α蛋白表达,证实转染成功,再采用噻唑蓝(MTT)比色法和Transwell小室体外侵袭实验检测转染后对U87细胞增殖体外侵袭能力的影响,流式细胞仪检测细胞凋亡.结果 转染靶向HIF-1α的短链发夹RNA的胶质瘤细胞,HIF-1α蛋白表达较空白细胞组明显降低,MTT法检测转染组、空载体组和空细胞组24 h细胞的增殖率分别为5.46%、21.25%、22.32%,体外侵袭性实验表明转染组、空载体组和空细胞组细胞12 h侵袭ECM的细胞数分别为(22±4)、(124±3)、(122±6);流式细胞仪检测表明转染组、空载体组和空细胞组细胞凋亡率分别为(53.35±2.80)%、(12.02±1.60)%、(10.19±3.15)%,转染组与空白细胞组及空载体组比较差异均有统计学意义(P＜0.05).结论 封闭HIF-1α蛋白的表达,可以抑制人胶质瘤U87细胞增殖和侵袭能力,促进细胞凋亡.

Abstract：

Objective To investigate the effects of antisense oligodeoxynucleotides (ODNs) targeting hypoxia inducible factor-1α (HIF-1α) on the apeptosis, proliferation and invasion of U87 glioma cell line. Methods Antisense ODNs were constructed and transfected into U87 cells by Dosper liposomal reagent. The HIF-1α gene expression was detected by Western blotting, the cell proliferative index was determined by methyl thiazol tetrazolium (MTT) assay, the cells cycle and apoptosis of the cells were examined by flow cytometry and the changes of the U87 cells invasive ability were measured by Transwell chamber.Results The protein expression of HIF-1α in U87 cells was down-regulated by HIF-1α ASONDN. The cell proliferative index in transfected group, empty vector group and control group was 5.46％, 21.25％and 22. 32％ respectively. Transwell chamber assay showed that the cell number in transfected group,empty vector group and control group was (22 ±4), ( 124 ±3) and ( 122 ±6) respectively; and the apoptosis rate was (53. 35 ± 2. 80) ％, ( 12.02 ± 1.60 )％, ( 10. 19 ± 3. 15 )％ respectively, and there was significant difference between transfected group and other groups ( P ＜ 0. 05 ). Conclusion Silencing the expression of HIF-1α protein can inhibit proliferation and invasion, and promote apoptosis of human glioma U87 cells. HIF-1α is expected to become a target for cancer therapy.     

高糖对骨形态发生蛋白-2、胰岛素样生长因子-1基因转染大鼠骨髓基质干细胞增殖的影响

目的 观察高糖环境下骨形态发生蛋白-2(BMP-2)和胰岛素样生长因子-1(IGF-1)基因转染大鼠骨髓基质干细胞(BMSC)后BMSC的增殖.方法 用Ad-BMP-2和Ad-IGF-1转染大鼠BMSC,Wester blot检测蛋白表达.噻唑蓝(MTT)比色法及流式细胞术检测细胞增殖.结果 Western blot检测到转染组细胞中有目的 蛋白表达.MTT结果显示第5天细胞增殖能力达到高峰,5 d光吸收值:A～E组分别为0.324±0.027、0.319±0.017、0.622±0.028、0.626±0.020、0.778±0.031.流式细胞术结果显示A～E组细胞处于增殖期的比重分别为23.92±3.07、23.51±2.11、34.37±6.85、35.04±1.45、42.56±1.15.结论 高糖环境下BMP-2和IGF-1基因转染均能促进BMSC增殖,联合转染对BMSC增殖有协同效应.

Abstract：

Objective To observe the proliferation of bone marrow stromal cell (BMSC) transfected by bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 ( IGF-1 ) gene under high glucose condition.Methods Ad-BMP-2 and Ad-IGF-1 transfected rat BMSC,protein expression of BMSC were detected by Western blotting analysis.Methyl thiazol tetrazolium (MTT) assay and flow cytometry to observe the proliferation potential of BMSC.Results In the Western blotting analysis,positive signal lane of protein was observed in transfected group.MTT assay show that proliferation reached the peak in all groups on the fifth day,and the absorbency values of A to E group were 0.324 ± 0.027,0.319 ± 0.017,0.622 ±0.028,0.626 ± 0.020,0.778 ± 0.031.Flow cytometry show that the proliferative percentage from A to E group were 23.92 ±3.07,23.51 ±2.11,34.37 ±6.85,35.04 ± 1.45,42.56 ± 1.15.Conclusion BMP-2 or IGF-1 can promote the proliferation of BMSC under high glucose condition,but the combined has the synergy effect.     

RNA干扰抑制CD44和CD133的表达对肺腺癌A549细胞增殖能力的影响

目的 观察CD44和CD133的表达对人肺腺癌A549细胞株增殖能力的影响.方法 筛选可持续增殖的A549细胞并培养,按CD44和133表达的不同将筛选出的细胞分成5组,非转染组:CD44+CD133+细胞(A组)、CD44-/CD133-细胞(B组)、未分选A549细胞(C组);转染组:CD44-和CD133-细胞(D组)及阴性对照组(E组).筛选得到沉默效果最佳的小干扰RNA(siRNA)片段,并用其转染各组细胞,采用MTS法检测并比较干扰前后各组细胞的增殖能力.结果　沉默效果最佳的siRNA片段及浓度为siCD44-h_002 25 nmol/L和siCD133-h_002 25 nmol/L,沉默效率分别为62%和82%.用其转染各组样本后,CD44和CD133的表达明显被抑制,比较转染前后各组样本的灰度值,差异有统计学意义(P＜0.01).通过分析MTS图显示,转染后细胞的增殖能力显著下降.结论　CD44和CD133的阳性表达,与人肺腺癌A549细胞株较强的增殖能力有关,CD44和CD133可能是肺腺癌干细胞的表面标志物.

Abstract：

Objective To study the effect of expression of CD44 and CD133 on the tumorigenesis of human lung adenocarcinoma cell line A549. Methods A549 cells having continuous self-renewal ability were cultured, and classified into 5 subpopulations based on differential expression patterns for CD133and CD44. The small interfering RNA (siRNA) with optimal sequences was screened out, and transfected the A549 cells. The methods of MTS and cell counting were used to investigate the proliferation of A549cells before and after the expression of CD133 and CD44 was inhibited by RNAi. Results For the optimal sequences and concentrations for siRNA transfection efficiency being 25 nmol/L for both siCD44-h_002 25and siCD133-h_002 25, the transfection efficiency was 62% and 82% respectively. Stably transfected cells were screened, and the expression of CD44 and CD133 was inhibited significantly. The gray values before transfection were significantly different from those after transfection ( P ＜ 0. 01 ). The MTS curve showed that the cell viability was significantly decreased after transfection. Conclusion The proliferation ability of A549 cells was related to the positive expression of CD44 and CD133. CD133 and CD44 may be important surface markers of lung cancer stem cells.     

The expression of TGF-βmRNA and its biological function in rat osteoblasts with exposure to raloxifene

Objective To investigate the effects of raloxifene and estradiol on the proliferation, differentiation and the expression of transforming growth factor-β(TGF-β) of osteoblasts in vitro.Methods Different doses of raloxifene and estradiol were added into the medium of the second generation osteoblasts obtained from the skull of newborn SD rats.The following parameters including cell proliferation,activity of alkaline phosphatase(ALP), the levels of type I collagen(Col-I) mRNA and TGF-β1 mRNA in different groups were measured and analyzed.Results Raloxifene and 17-βestradiol(17-β E2 ) showed no significant effect on stimulating the proliferation of osteoblasts (P＞0.05 vs the control).However, raloxifene could significantly improve ALP activity and Col-I mRNA expression in high consistency group (P＜0.01) in dose-dependent manner.Raloxifene group in 10-8 mol/L increased the expression of TGF-β1 mRNA (vs the control, P ＜ 0.05).Estradiol significantly increased ALPactivity, Col-I mRNA expression and TGF-β1 mRNA expression (P＜0.05 or P＜0.01 vs the control).Conclusions Both of raloxifene and estradiol could stimulate differentiation of osteoblasts and expression of bone matrix, but showed no effect on proliferation of cultured osteoblasts.The expressions of TGF-β1 mRNA were different, which might imply their different mechanisms by means of estrogen receptor.     

The expression of TGF-βmRNA and its biological function in rat osteoblasts with exposure to raloxifene

Objective:To investigate the effects of raloxifene and estradiol on the proliferation,differentiation and the expression of transforming growth factor-β(TGF-β)of osteoblasts in vitro.Methods:Different doses of raloxifene and estradiol were added into the medium of the second generation osteoblasts obtained from the skull of newborn SD rats.The following parameters including cell proliferation,activity of alkaline phosphatase(ALP),the levels of type Ⅰ collagen(Col-I)mRNA and TGF-β1 mRNA in different groups were measured and analyzed.Results:Raloxifene and 17-βestradiol(17-β E2)showed no significant effect on stimulating the proliferation of osteoblasts(P>0.05 vs the control).However,raloxifene could significantly improve ALP activity and Col-I mRNA expression in high consistency group(P<0.01)in dose-dependent manner.Raloxifene group in 10-8 mol/L increased the expression of TGF-β1 mRNA(vs the control,P<0.05).Estradiol significantly increased ALP activity,Col-I mRNA expression and TGF-β1 mRNA expression(P<0.05 or P<0.01 vs the control).Conclusions:Both of raloxifene and estradiol could stimulate differentiation of osteoblasts and expression of bone matrix,but showed no effect on proliferation of cultured osteoblasts.The expressions of TGF-β1 mRNA were different,which might imply their different mechanisms by means of estrogen receptor.     

肺癌相关基因SLC35F2 RNA干扰重组慢病毒载体的构建和鉴定

目的 针对肺癌相关基因SLC35F2构建RNA干扰(RNAi)重组慢病毒质粒并进行慢病毒包装,建立SLC35F2表达稳定抑制的H1299肺癌细胞株,并探讨SLC35F2基因的功能.方法 应用pGCSIL-PUR慢病毒载体构建针对SLC35F2的ShRNA载体,转染包装293T细胞,收集病毒上清转染肺癌细胞株H1299,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量聚合酶链反应(Real-time PCR)和Western blot检测癌细胞内SLC35F2的表达;CCK-8比色法检测细胞增殖;Annexin V-FITC/PI双染法检测细胞凋亡.结果 成功构建SLC35F2-ShRNA慢病毒载体(SLC-siRNA)及SLC35F2表达稳定抑制的肺癌细胞株,经过检测证实SLC-siRNA慢病毒载体对SLC35F2的抑制效率达81.8%;CCK-8检测显示SLC-siRNA稳定转染的H1299细胞株生长抑制率达16.3%,稳定转染株凋亡细胞百分比较阴性对照组明显升高(14.88%比3.16%,P＜0.05).结论 慢病毒载体介导的靶向SLC35F2的RNAi可有效抑制SLC35F2表达,降低肺癌细胞的增殖能力,增加其凋亡比例.

Abstract：

Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference (RNAi) and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 (SLC-siRNA) was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction (PCR) and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group ( 14.88% vs 3.16% ,P ＜0.05 ).Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established.     

抑制骨桥蛋白表达降低肺癌侵袭和增殖的机制

目的 探讨抑制骨桥蛋白(OPN)表达降低肺癌细胞株A549侵袭、增殖的机制.方法 构建针对人OPN mRNA干扰质粒pENTRTM/U6-INF(pINF-1)及对照质粒pENTRTM/U6-CTR(pCTR),将其转染A549细胞,Western blot测定OPN、基质金属蛋白酶(MMP)和促分裂素原活化蛋白激酶(MAPK)信号通路相关蛋白的表达;明胶酶谱检测MMP的表达.结果 与空白对照组(1.20±0.15)比较,转染72 h后pINF-1组OPN蛋白表达(0.15±0.04)下降87%,与对照组比差异有统计学意义(P＜0.05).Western blot的结果显示,pINF-1组细胞磷酸化细胞外信号调节激酶1/2(pERK1/2)、磷酸化丝裂原细胞外激酶(pMEK)和MMP-2的表达明显下降,差异有统计学意义(P＜0.01);而MMP-9表达差异无统计学意义(P＞0.05).明胶酶谱结果同样表明pINF-1组MMP-2的表达明显下降(P＜0.01).结论 抑制OPN的表达降低肺癌细胞株A549侵袭力和增殖的机制可能与抑制MAPK信号通路和MMP-2的表达有关.

Abstract：

Objective To explore the mechanism of invasion and proliferation of lung cancer cell line A549 mediated by osteopontin. Methods One double-stranded DNA vectors pENTRTM/U6-INF ( pINF-1 ) targeting the mRNA of human osteopontin ( OPN), and the control vector pENTRTM/U6-CTR (pCTR) mismatching with mRNA of OPN were constructed, and then they were transfected into human A549 cells with high metastatic potentials. Western blotting was used to quantify the protein level of OPN,matrix metalloproteinases (MMPs) and mitogen-activated protein kinases (MAPK). The activity of MMPs was detected by gelatin zymography. Parallel experiments were performed in sextuplicate. Results As compared with control group only transfected with LipofectamineTM 2000, OPN protein level in pINF-1 group was decreased by 87% 72 h after transfection (P ＜0. 05), and no significant difference was found in the group transfected with pCTR. Moreover, the expression levels of phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), phosphorylated MAPK/ERK1/2 kinase (pMEK) and MMP-2 protein were also decreased in pINF-1 group (P ＜ 0. 01 ). The activity of MMP-2 but not MMP-9 was decreased significantly in pINF-1 group (P ＜ 0. 01 ). Conclusion OPN plays an important role in metastasis as well as tumor growth of lung cancer cells probably through activation of the MAPK pathways and MMP-2.     

小分子干扰RNA降低蛋白激酶Cι表达抑制HeLa细胞增殖

目的 观察小分子干扰RNA(siRNA)沉默蛋白激酶Cι (PKCι)表达对HeLa增殖与凋亡的影响.方法 实验分为转染组、阴性对照和空白对照组.将PKCι基因的siRNA转染HeLa细胞,噻唑蓝(MTT)比色法检测3组细胞第1～5天的吸光度;培养细胞于第21天计数细胞克隆数;流式细胞仪检测沉默PKCι基因72h的细胞凋亡及细胞周期分布.结果 转染组细胞增殖率低于两个对照组.转染组的平板集落形成率(64.00±4.54)%低于阴性(87.00±1.22)%和空白组(82.00±2.10)%(P<0.05).转染组细胞凋亡率(9.54±0.55)%明显高于阴性对照组(1.31±0.10)%和空白对照组(2.75±0.24)%.瞬时转染PKCι siRNA 72h后,G1期细胞增多,G2+S期细胞减少.结论 沉默PKCι可抑制宫颈癌细胞增殖,促进细胞凋亡.

Abstract：

Objective To explore the effect of RNA interference (RNAi) targeting protein kinase C iota (PKCι) on proliferation and apoptosis of cervical carcinoma cell line HeLa. Methods Stable cell lines which were transfected with PKCι shRNA or vector-mock plasmids were established. The cell lines, psi-PKCiota-HeLa, psi-mock-HeLa and HeLa, were used to detect methyl thiazol tetrazolium (MTT) absorbance at 1st-5th day. The cells were cultured in 6-well plates and stained by crystal violent at 21st day, and the stained cells colonies were counted. Flow cytometry was used to detect apoptosis and cell cycle distribution after HeLa cells were transiently transfected with PKC iota shRNA at 72nd h. Results Compared with the psi-mock and blank controls, the transfected cells stably expressing PKCι shRNA showed markedly decrease of proliferation assayed by MTT and plate colony formation. The apoptosis rate of cells transiently transfected with PKCι shRNA (9.54±0.55)% was higher than that of cells transiently transfected with psi-mock (1.31±0.10)% or HeLa blank control cells (2.75±0.24)% (P<0.05). The cells transiently transfected by PKCι shRNA were arrested in G0/G1 phase. Conclusion PKCι is essential for maintaining of HeLa cell proliferation. Silencing PKCι can inhibit the growth of HeLa cells and induce apoptosis.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.