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.     

消炎痛抑制肝细胞肝癌生长转移的研究

Objective To study the effects of indomethacin on proliferation and invasion of hepatocellular carcinoma (HCC) cell line MHCC97L with metastatic potential and the effect of indomethacin on the growth and metastasis of HCC. Methods (1) In vitro; Proliferation, Transwell invasion assay, cell motility assay, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein activity were evaluated after cells were treated with 0. 2 mmol/L indomethacin. (2)In vivo: Mice bearing xenografts in the liver were randomly divided into control and indomethacin groups. At the end of sixth week, the mice were killed and tumor volume, inhibitory rate, immunohistochemistry assay (IHA) and metastasis were evaluated. Results (1)In vitro; 0. 2 mmol/L indomethacin could inhibit the proliferation of MHCC97L cells markedly (P ＜0. 01). The average amount of invading cells per field in cell invasion assay and motility assay was 2. 2 ± 1. 3 and 4.4 ± 1. 1 respectively in indomethacin group, significantly less than in control group ( 11. 4 ± 1. 9 and 12. 8 ± 1. 8 respectively, P ＜0. 01). The expression of VEGF and MMP-2 in cells treated with indomethacin was significantly lower than in control group (P ＜0. 01). (2)In vivo; Tumor volume, incidence and number of lung metastases in control and indomethacin groups were (1700 ±422) mm3 and (1170 ± 585) mm3 (P ＜ 0. 05), 75% and 50% ( P ＞ 0.05), 2. 92 ± 2. 07 and 1.33 ±1.56 (P＜0. 05) , respectively. Inhibition rate in indomethacin group was 31.2%. IHA showed that the expression of VEGF, MMP-2, and cyclooxygenase-2 ( COX-2) was down-regulated in indomethacin group (P ＜0.01). Conclusion Indomethacin could inhibit the growth and metastasis of HCC, which was in part mediated by down-regulation of VEGF and MMP-2.     

消炎痛抑制肝细胞肝癌生长转移的研究

Objective To study the effects of indomethacin on proliferation and invasion of hepatocellular carcinoma (HCC) cell line MHCC97L with metastatic potential and the effect of indomethacin on the growth and metastasis of HCC. Methods (1) In vitro; Proliferation, Transwell invasion assay, cell motility assay, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein activity were evaluated after cells were treated with 0. 2 mmol/L indomethacin. (2)In vivo: Mice bearing xenografts in the liver were randomly divided into control and indomethacin groups. At the end of sixth week, the mice were killed and tumor volume, inhibitory rate, immunohistochemistry assay (IHA) and metastasis were evaluated. Results (1)In vitro; 0. 2 mmol/L indomethacin could inhibit the proliferation of MHCC97L cells markedly (P ＜0. 01). The average amount of invading cells per field in cell invasion assay and motility assay was 2. 2 ± 1. 3 and 4.4 ± 1. 1 respectively in indomethacin group, significantly less than in control group ( 11. 4 ± 1. 9 and 12. 8 ± 1. 8 respectively, P ＜0. 01). The expression of VEGF and MMP-2 in cells treated with indomethacin was significantly lower than in control group (P ＜0. 01). (2)In vivo; Tumor volume, incidence and number of lung metastases in control and indomethacin groups were (1700 ±422) mm3 and (1170 ± 585) mm3 (P ＜ 0. 05), 75% and 50% ( P ＞ 0.05), 2. 92 ± 2. 07 and 1.33 ±1.56 (P＜0. 05) , respectively. Inhibition rate in indomethacin group was 31.2%. IHA showed that the expression of VEGF, MMP-2, and cyclooxygenase-2 ( COX-2) was down-regulated in indomethacin group (P ＜0.01). Conclusion Indomethacin could inhibit the growth and metastasis of HCC, which was in part mediated by down-regulation of VEGF and MMP-2.     

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation,and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.     

聚酰胺-氨纳米微粒介导5-氟尿嘧啶联合小RNA-21抑制乳腺癌细胞生长的研究

Objective To study the effect of PAMAM-mediated 5-fluorouracil combined with miR-21 inhibitor gene therapy to suppress MCF-7 human breast cancer cell growth in vitro. Methods 5-Fu/PAMAM complex was prepared by dialysis method and then incubated with miR-21 inhibitor at room temperature. Transmission electronic microscopy (TEM) was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy (UV). The transfection of PAMAM dendrimer was detected by flow cytometry. MTT assay was carried out to determine MCF-7 cell growth survival rate. Cell apoptosis was analyzed by flow-cytometry. Transwell assay was performed to detect invasion ability after MCF-7 cells treated with 5-Fu chemotherapy combined with miR-21 inhibitor gene therapy. Results The morphology of the complex was sphere observed under TEM. Encapsulation efficiency and loading efficiency of drug were (66. 21±4. 11)% and (31.77±0. 73)% , respectively. Flow cytometry revealed that 5-Fu/PAMAM transfection efficiency was (60.54 ±6. 97)%. 5-Fu combined with miR-21 inhibitor treatment significantly suppressed cell growth, and the survival rate was only (55. 85±3. 71)% on the 6th day of the observation period. The apoptosis rate in combined treatment group was (18. 32±2.42)% , dramatically higher than in control group (F=58. 326,P<0. 01). In combined treatment group, the number of invasion cells was only 18. 96 ±3. 14, suggesting the greatly decreased invasion ability of MCF-7 cells (F=16. 409,P < 0. 01). Conclusion PAMAM could effectively deliver miR-21 inhibitor and 5-Fu simultaneously, and combined therapy can suppress growth of MCF-7 cells effectively in vitro.     

聚酰胺-氨纳米微粒介导5-氟尿嘧啶联合小RNA-21抑制乳腺癌细胞生长的研究

Objective To study the effect of PAMAM-mediated 5-fluorouracil combined with miR-21 inhibitor gene therapy to suppress MCF-7 human breast cancer cell growth in vitro. Methods 5-Fu/PAMAM complex was prepared by dialysis method and then incubated with miR-21 inhibitor at room temperature. Transmission electronic microscopy (TEM) was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy (UV). The transfection of PAMAM dendrimer was detected by flow cytometry. MTT assay was carried out to determine MCF-7 cell growth survival rate. Cell apoptosis was analyzed by flow-cytometry. Transwell assay was performed to detect invasion ability after MCF-7 cells treated with 5-Fu chemotherapy combined with miR-21 inhibitor gene therapy. Results The morphology of the complex was sphere observed under TEM. Encapsulation efficiency and loading efficiency of drug were (66. 21±4. 11)% and (31.77±0. 73)% , respectively. Flow cytometry revealed that 5-Fu/PAMAM transfection efficiency was (60.54 ±6. 97)%. 5-Fu combined with miR-21 inhibitor treatment significantly suppressed cell growth, and the survival rate was only (55. 85±3. 71)% on the 6th day of the observation period. The apoptosis rate in combined treatment group was (18. 32±2.42)% , dramatically higher than in control group (F=58. 326,P<0. 01). In combined treatment group, the number of invasion cells was only 18. 96 ±3. 14, suggesting the greatly decreased invasion ability of MCF-7 cells (F=16. 409,P < 0. 01). Conclusion PAMAM could effectively deliver miR-21 inhibitor and 5-Fu simultaneously, and combined therapy can suppress growth of MCF-7 cells effectively in vitro.     

RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

Objective To discuss the effects of silencing of iASPP gene on human bladder cancer cells. Methods RNAi silencing of iASPP gene in bladder cancer cell 5637 and T24 cells were used by lentiviral mediated interfering short hairpin RNAs. Cell proliferation was tested by MTT assay, and rate of colony was tested by colony formation assay. Cell cycles were tested by using fluorescence-activated cell sorting. Results Down-regulation of iASPP could inhibit the growth and proliferation of human bladder cancer cells (P＜0.05). iASPP know-down could decrease the colony formation of 5637 and T24 cells (P＜0, 05). Knocking down of iASPP in 5637 and T24 cells showed cell arrested at G1. Conclusions Silencing of iASPP gene could inhibit proliferation and colony formation of bladder cancer, iASPP might be an important target for gene therapy of bladder cancer.     

抗肝肠钙粘连蛋白单克隆抗体对肝癌细胞生物学行为的影响

目的 观察抗肝肠钙粘连蛋白(CDH17)单克隆抗体Lic5对肝癌细胞生物学行为的影响.方法 Western blot和实时定量聚合酶链反应(PCR)检测细胞株MHCC97H、MHCC97L、PLC/PRF/5及MIHA中CDH17的表达.噻唑蓝(MTT)比色法、细胞划痕法、Transwell法及平板克隆法检测Lic5对肝癌细胞生物学行为的影响.结果 CDH17仅在细胞株MHCC97H、MHCC97L中表达,Lic5可结合肝癌细胞表面的CDH17,并抑制CDH17表达.Lic5 50mg/L组、100mg/L组、小鼠IgG组4 d细胞增殖抑制率在MHCC97H为26.1%、43.6%、6.4%,MHCC97L为26.0%、40.7%、7.7%;Lic5100mg/L组、小鼠IgG组、磷酸盐缓冲液(PBS)组48h细胞迁移抑制率在MHCC97H为36.7%、8.4%、5.6%,MHCC97L为42.3%、10.2%、7.4%(P＜0.05);穿膜细胞数在MHCC97H为(39.20±9.56)、(106.50±7.56)、(96.60±13.02)个,MHCC97L为(26.00±8.61)、(86.00±10.26)、(90.40±12.04)(P＜0.05);克隆形成数在MHCC97H为(59.30±11.68)、(141.70±19.40)、(150.30±14.64),MHCC97L为(57.20±10.21)、(132.50±9.07)、(121.70±11.93)(P＜0.01).Lic5对PLC/PRF/5及MIHA细胞的生物学行为无明显影响.结论 单克隆抗体Lic5能够下调肝癌细胞CDH17表达,抑制肝癌细胞的增殖、迁移、侵袭和克隆形成能力.

Abstract：

Objective To investigate the effect of monoclonal antibody against liver-intestine cadherin (CDH17) on the cell proliferation, migration, invasion and colony formation of hepatocellular carcinoma cell lines. Methods Hepatocellular carcinoma cell lines MHCC97H, MHCC97L, PLC/PRF/5 and MIHA were examined for CDH17 expression by Western blotting and quantitative real-time polymerase chain reaction (PGR). The combination capacity between bepatocellular carcinoma cell lines and monoclonal antibody Lic5 was detected by the way of immunofluorescence staining. The cell lines were treated with Lic5, PBS and mouse IgG respectively. Methyl thiazol tetrazolium (MTT) assay, wound healing assay, Transwell invasion assay and colony formation assay were used to study the changes in cell proliferation, migration, invasion and colony formation of hepatocellular carcinoma cell lines. Results High expression level of CDH17 was detected in MHCC97H and MHCC97L cell lines. CDH17 protein level was down-regulated but there was no significant effect on CDH17 mRNA after treatment with Lie5 in MHCC97H and MHCC97L. Cellular growth rate of MHCC97H in Lic5 (50 mg/L), Lic5 ( 100 mg/L) and mouse IgG groups was decreased by 26. 1%, 43.6% and 6. 4%, and by 26. 0%, 40. 7% and 7. 7% in MHCC97L on the 4th day respectively (P ＜0. 05 ). The inhibition rate of cell migration at 48 h was 36. 7%, 8. 4% and 5.6% in Lic5 ( 100 mg/L), mouse IgG and PBS groups in MHCC97H, and 42. 3%, 10. 2% and 7. 4% in MHCC97L respectively ( P ＜ 0. 05 ). The number of invasion cells was ( 39. 20 t 9. 56),(106.50±7.56) and (96.60±13.02) in MHCC97H, and (26.00±8.61), (86.00±10.26) and (90.40±12.04) in MHCC97L in Lic5 (50 mg/L), Lic5 (100 mg/L) and mouse IgG groups, respectively (P ＜ 0. 05 ). The number of colony formation was ( 59. 30 ± 11.68 ), ( 141.70 ± 19. 40 ) and (150.30 ±14.64) in MHCC97H, and (57.20 ± 10.21), (132.50 ±9.07) and (121.70 ±11.93) in MHCC97L in Lie5 (50 mg/L), Lic5 (100 mg/L) and mouse IgG groups, respectively (P＜ 0. 01 ).There was no significant difference between Lic5 treatment groups and controls in PLC/PRF/5 and MIHA cell lines. Conclusion The anti-CDH17 monoclonal antibody Lic5 can down-regulate CDH17 expression and inhibit the cell proliferation, migration, invasion and colony formation in hepatocellular carcinoma cell lines.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

膝关节后交叉韧带断裂治疗临床分析

目的 对35例膝关节后交叉韧带断裂治疗进行临床分析,重点探讨了有关交叉韧带断裂的治疗问题。方法 经明确诊断后,分析采用胫骨附着处撕脱骨折复位固定手术治疗26例、早期髌韧带中1／3移植重建3例、单纯长腿石膏固定6例。结果 本组病例全部进行随访,随访时间13个月－5年,胫骨附着处撕脱骨折复位固定及髋韧带中1／3移植重建29例为优良、单纯长腿石膏固定6例为差。结论 后交叉韧带断裂后应该及时给予手术修复;膝后外侧手术入路,操作简单,暴露充分;少于3个月的陈旧性病例仍适应手术治疗。     

不同方法重建指尖离断静脉回流的疗效分析

目的：探讨不同方法重建指尖离断静脉回流的疗效。方法：2008年3月-2013年2月收治指尖离断患者80例,38例吻合指侧方静脉重建回流,术中吻合动静脉比例1：1或1：2或2：2,平均1：2;22例吻合指腹静脉重建回流,术中吻合动静脉比例1：1;20例未吻合静脉,术中仅吻合1条动脉,行侧切口或甲床放血。观察各组治疗效果。结果：吻合指侧方静脉组手指全部成活,无一例发生回流障碍;吻合指腹静脉组19例发生静脉危象,其中4例手指坏死;未吻合静脉组20例均发生回流障碍,其中6例手指坏死。58例获随访,随访时间6~28个月。吻合指侧方静脉组32例,指尖外形佳、指腹饱满;吻合指腹静脉组14例,指体轻度萎缩,指甲生长不平整;未吻合静脉组12例,指体萎缩明显。吻合指侧方静脉组指甲生长近平整,长度长于其他两组[（14.4±3.2）mm比（12.5±2.3）mm和（12.2±2.2）mm],远侧指间关节活动度大于其他两组[（63±5）°比（48±3）°和（45±7）°],两点分辨觉小于其他两组[（4.6±0.4）mm比（7.1±1.2）mm和（7.3±0.6）mm],感觉级别高于其他两组[S（3.45±0.39）级比S（2.57±0.42）级和S（2.55±0.49）级],差异均具有显著性（P〈0.05）。吻合指腹静脉组和未吻合静脉组在指甲长度、运动和感觉方面差异无统计学意义（P〉0.05）。结论：吻合指侧方静脉能有效解决指尖再植静脉回流问题,可避免回流障碍,成活率高,促进指甲生长,可恢复 DIPJ 活动度及感觉。     

胫骨干骨折的手术治疗对策

目的：明确不同固定器械在胫骨干不同骨折类型固定中的特点，以指导临床应用。方法：68例胫骨干骨折，行加压钢板螺钉、交锁髓内钉、单侧外固定架固定后，作临床疗效分析。结果：加压钢板固定组42例，感染5例，骨不连1例，平均愈合时间3．8个月；交锁髓内钉固定组13例，无感染及骨不连，平均愈合时间5．4个月；单侧外固定架组13例，骨不连1例，踝关节背伸受限3例，平均愈合时间4．5个月。结论：胫骨骨折交锁髓内钉固定并发症少，功能恢复好，适用范围广，但要注意及时进行动力加压。加压钢板及外固定架固定应选择各自的最佳适应证，以达到理想的疗效。