目的 合成可生物降解的基因载体，并分析其生物毒性及转染率。 方法 低分子量聚乙烯亚胺（PEI）通过双硫键交联合成可降解的高分子量PEI衍生物（SS-PEI），通过红外光谱和核磁波谱分析技术分析其化学结构，采用细胞活力实验和检测大鼠肝肾功能指标分析其细胞和体内毒性，并转染羟基荧光素修饰的siRNA（FAM-siRNA）分析细胞转染率。 结果 红外波谱和核磁波谱分析可见酰胺键的特征波谱，噻唑蓝法和肝肾功能指标显示SS-PEI不同剂量组与对照组的差异均无统计学意义（P＞0.05），SS-PEI/FAM-siRNA转染率为（76.0 ± 2.8）%。 结论 SS-PEI的合成可明显提高装载siRNA的效率，具有安全、高效等特点。
Objective To observe the effect of RNA interference (RNAi) on HepG2 hepatic cancer cell by small interfering RNA (siRNA). Methods siRNA targeting vascular endothelial growth factor (VEGF) gene was transfected into HepG2 cells by LipofectimineTM 2000. The VEGF mRNA and protein were respectively detected by real-time quantitive PCR and Western blot, and the concentration of VEGF protein in the cell culture supertant was determined by ELISA at 48 h after culture. Results The average efficiency of siRNA transfection was (90.4±2.9)% after 6 h cell culture. The expressions of VEGF mRNA and protein in HepG2 cells could be effectively suppressed by siRNA, and the concentration of VEGF protein in the cell culture supertant was also decreased. Conclusion siRNA can knock down the expression of VEGF gene and decrease the concentration of VEGF protein in HepG2 cells.
Objective To construct small interfering RNA（siRNA） eukaryotic expression vector specific for human hnRNP K gene，and to observe its silencing effects on hnRNP K gene in A549 cells.Methods The expression vectors of pSUPER/hnRNP K siRNAa，pSUPER/hnRNP K siRNAc and pSUPER/siRNAn were constructed by gene recombination and then transfected into the A549 lung carcinoma cell line by using Lipofectamine2000（a and c respectively represented A and C fragments in hnRNP K coding sequence contained 19 nts，n represented nonsense fragment as control）.The mRNA and protein were harvested after 24 h and analyzed for the expression of hnRNP K by RT-PCR and Western blotting respectively.Results The siRNA vector targeted to hnRNP K successfully decreased hnRNP K mRNA and protein levels 24 h after transfection in A549 cells.Relative expressed doses of hnRNP K mRNA in lung cancer cells transfected by hnRNP K siRNAa and hnRNP K siRNAc respectively were 0.24±0.53 and 0.28±0.57 after 24 h，which were significantly lower than that in the control group（both Plt;0.01）.The gray scale values of hnRNP K protein were 0.23±0.11 and 0.28±0.09 respectively，which were also significantly lower than those in the control group（both Plt;0.05）.And pSUPER/hnRNP K siRNAa was the most effective one.Conclusion Eukaryotic expression vector of siRNA specific for hnRNP K is successfully constructed，which lays the basis for the function study of hnRNP K gene and its application in the treatment of lung carcinoma.
Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α). Methods The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05). Conclusion The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.
【Abstract】ObjectiveTo investigate the effect of RNA editing enzyme ADAR1 on the function of lymphocyte immune by transferring mouse lymphocytes with plasmid of sense siRNA and by suppressing the expression of ADAR1. MethodsThe cell strains of human hepatic cellular carcinoma （HCC） were frozen and thawed repeatedly to prepare for tumor soluble antigen. The isolated mouse lymphocytes, which were transferred with antisense siRNA plasmid of ADAR1 and were sensitized with soluble tumor antigen were used as the study group; those which were not transferred but were sensitized were used as the control group. The 3HTdR adulteration experiment was used to test the sensitivity of lymphocytes. The effect of ADAR1 on lymphocyte immunity was detected by lymphocytotoxicity tests. ResultsThe observation of the isolated lymphocytes implied that the growth cycle of lymphocyte was 10-14 days. The 3HTdR adulteration experiment showed the result was optimal. The number of HCCs decreased significantly for both of the groups compared with those in the blank holes, but the amplitude was much larger in the control group. The expression of ADAR1 in lymphocytes of the study group was significantly lower than that of the control group, which demonstrated that the RNA plasmid of ADAR1 suppressed the expression of ADAR1 in sensitized lymphocytes and the suppressing rate of the control group (87.47±4.62)% was significantly higher than that of study group （53.19±3.95）%. The function of lymphocytes killing targetcells in the study group was significantly inferior to that of control group (P＜0.05). ConclusionRNA editing enzyme ADAR1 may play an important role in mouse cellullar immunologic response and it is possible to attenuate the cellimmune response by depressing the expression of ADAR1.
Objective By using small interfering RNAs ( siRNAs) specific for spleen tyrosine kinase ( Syk) , to evaluate the role of Syk in maturation of bone marrow-derived dendritic cells. Methods The fragments of 21-23 bp siRNAs specific for mice Syk were chemo synthesized and transfected into the asthmatic murine bone marrow-derived dendritic cells ( BMDCs) by Lipofectamine 2000 transfection system for 48 hours. Then BMDCs were co-cultured with T cells from the normal mice spleen for 48 hours. The cytokines including IL-4, IL-13, IL-2 and INF-γin supernatant were detect by ELISA. The expression of Syk protein was measured by Western Blot to determine whether the Syk gene was silenced. Results The expression of Syk protein was obviously decreased in the siRNA-interference group. The secretions of IL-4 and IL-13 were significantly inhibited by siRNA interference ( P lt; 0. 05) , but the secretions of IL-2 and INF-γwere not interfered signficantly ( P gt;0. 05) . Conclusion Syk specific siRNA fragments can block the antigen presentation function of dendritic cells and block the activation and differentiation of T cells.
目的了解载体携带的小干扰RNA（small interfering RNA, siRNA）对人胰腺癌细胞株PANC-1血管内皮生长因子（vascular endothelial growth factor, VEGF）表达的抑制作用以及对肿瘤细胞生长的影响。方法 设计并合成2对针对VEGF的siRNA 真核表达载体（PU-VEGF-siRNA1 组和 PU-VEGF-siRNA2 组，简称为重组质粒组），经脂质体Lipofectamine 2000 转染PANC-1 细胞，经G418 筛选后获得稳定转染细胞株，空载体转染组作为实验对照组，未转染组作为空白对照组。采用MTT法检测转染后PANC-1 细胞的增殖，流式细胞仪检测转染后PANC-1 细胞的凋亡率和细胞周期，逆转录聚合酶链反应（RT-PCR）检测转染后VEGF mRNA 表达情况。结果 与实验对照组和空白对照组比较，两重组质粒组PANC-1 细胞的增殖减慢，细胞凋亡率增加，VEGF mRNA 表达明显下降，差异均有统计学意义（Plt;0.05）； 细胞周期结果显示，两重组质粒组S 期细胞所占比例较两对照组明显减少（Plt;0.05）。 结论siRNA 能有效抑制胰腺癌细胞VEGF 的表达，并能在体外抑制人胰腺癌细胞株PANC-1 细胞的生长。
目的 观察下调Ras同源类似物E (RhoE)表达对人乳腺癌细胞231生物学行为的影响。 方法 蛋白质印迹技术检测小干扰RNA（siRNA）转染前后RhoE在乳腺癌细胞231中的表达；RhoE siRNA的细胞转染 用lipofectamine?2000脂质体法；Cell Counting Kit-8检测转染细胞及对照细胞的增殖变化；损伤刮擦试验和体外侵袭实验（Transwell小室）分别检测转染细胞及对照细胞的迁移与侵袭能力。 结果 RhoE在乳腺癌细胞231中的表达较高；成功转染RhoE siRNA的乳腺癌细胞，蛋白质印迹显示RhoE的表达被明显的抑制；RhoE的表达被抑制后对乳腺癌细胞的增殖、迁移和侵袭有着明显的促进作用。 结论 下调RhoE 表达能够明显促进乳腺癌细胞的增殖﹑迁移和侵袭，RhoE可能在乳腺癌的发生发展中起着重要作用。
Objective To explore the effect of Tie-2 small interference RNA (siRNA) treatment in human hepatoma transplanted subcutaneously in nude mice. Methods Tumor cells were implanted in the hind flank of male nude mice of 6 weeks. Tumor-bearing mice were divided into two groups （gene therapy group and control group） and injected intra-tumorally with Tie-2-siRNA/Lipofectamine and saline/Lipofectamine respectively. The tumor volume and weight, serum AFP and microvessel density (MVD) and the histological change of the tumor were tested after gene therapy. Results The growth inhibitory rates in gene therapy group were 26.94%， 53.01% and 68.91% on day 4， 7 and 10 after gene therapy respectively. The tumor volumes of gene therapy group (118.47, 111.57 and 104.59 mm3) were smaller than those of the control group (162.17, 237.46 and 336.41 mm3) respectively (P＜0.01), and the weight of tumor in gene therapy group was lighter than that of the control group 〔(0.89±0.09) g vs (1.24±0.03), P＜0.01〕. The AFP value in gene therapy group was obviously lower than that of the control group 〔(107.66±24.13) ng/ml vs (266.08±50.96) ng/ml, P＜0.01〕. There was significant diference of MVD between the gene therapy group (34.63±4.07) and the control group (81.01±9.44) with the method of immunohistochemitry (P＜0.01). Histopathology in the control group showed that the tumor volumes were bigger, and a high atypic of tumor cells were seen. The main pathological changes in tumor tissue of gene therapy group were necrosis, there were massive necrosis. The apoptosis cells were seen in the both of necrosis and non-necrosis areas in only 2 mice of gene therapy group. Conclusion Tie-2-siRNA inhibits the tumor growth and tumor angiogenesis， and is a possible new approach for liver neoplasm gene therapy.