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.
目的 采用RNA干扰技术沉默CCS（copper chaperone for SOD1）基因，构建相关小干扰RNA（siRNA），探索出针对CCS的高效siRNA序列。 方法 合成用于人脐静脉内皮细胞（HUVEC）细胞中沉默CCS基因的siRNA。应用脂质体转染的方法在HUVEC细胞中对CCS基因进行RNA沉默。蛋白免疫印迹Western blotting检测沉默前后CCS蛋白表达变化的情况，甲基四唑蓝法MTT检测转染前后细胞活力。最后用单因素方差分析对数据进行统计学分析，以确定有效的siRNA序列。 结果 转染前后细胞形态无肉眼可见变化，转染后细胞活力分别为98.5%和98.8%。CCS蛋白沉默率分别为63.7%和61.4%。 结论 采用siCCS-2和siCCS-3序列转染条件对HUVEC细胞活力损伤小，CCS沉默效率高，实验条件稳定，重复性好。为我们继续研究沉默CCS后抑制血管内皮细胞的生长增殖、血管形成提供了稳定的实验基础。
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.
ObjectiveTo investigate effect of small interfering RNA (siRNA) targeting inhibition of growth hormone receptor (GHR) on proliferation and invasion of human liver cancer line SMMC-7721. MethodsSMMC-7721 cells were transfected with siRNA targeting human GHR by GenMuteTM transfection regent.The cells were divided into three groups:blank control group (non-transfected siRNA),negative control group (transfected with non-specific siRNA),and specificity transfected group (transfected with expression specifically interfere by GHR siRNA).the relative expression of GHR mRNA was detected by using real-time PCR.the expression of GHR protein was detected by using Western blot.The cell proliferation was assessed by CCK-8 assay.And the ability of invasion was examined by Transwell assay. ResultsThe expressions of GHR mRNA and GHR protein in the specificity transfected group were significantly lower than those in the blank control group (P<0.05) and the negative control group (P<0.05).Compared with the blank control group and the negative control group,the absorbance value and the number of migrating cells of SMMC-7721 cells were decreased obviously (P<0.05) in the specificity transfected group. ConclusionsiRNA targeting human GHR could reduce capability of proliferation migration and invasion of SMMC-7721 cells.
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.
【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.
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。
Multidrug resistance (MDR) remains the major obstacle to the success of clinical cancer chemotherapy. P-glycoprotein (P-gp), encoded by the MDR1, is an important part with complex mechanisms associated with the MDR. In order to overcome the MDR of tumors, we in the present experimental design incorporated small interfering RNA (siRNA) targeting MDR1 gene and anticancer drug paclitaxel (PTX) into the solid lipid nanoparticles (SLNs) to achieve the combinational therapeutic effects of genetherapy and chemotherapy. In this study, siRNA-PTX-SLNs were successfully prepared. The cytotoxicity of blank SLNs and siRNA-PTX-SLNs in MCF-7 cells and MCF-7/ADR cells were detected by MTT; and the uptake efficiency of PTX in MCF-7/ADR cells were detected via HPLC method; quantitative real-time PCR and flow cytometry were performed to investigate the silencing effect of siRNA-PTX-SLNs on MDR1 gene in MCF-7/ADR cells. The results showed that PTX loaded SLNs could significantly inhibit the growth of tumor cells, and more importantly, the MDR tumor cells treated with siRNA-PTX-SLNs showed the lowest viability. HPLC study showed that SLNs could enhance the cellular uptake for PTX. Meanwhile, siRNA delivered by SLNs significantly decreased the P-gp expression in MDR tumor cells, thus increased the cellular accumulation of rhodamine123 as a P-gp substrate. In conclusion, the MDR1 gene could be silenced by siRNA-PTX-SLNs, which could promote the growth inhibition efficiency of PTX on tumor cells, leading to synergetic effect on MDR tumor therapy.
This study aims to investigate the effects of small interference RNA (siRNA) targeting PML-RARα mRNA on the activity of the acute promyelocytic leukemia cell line NB4. The proliferation inhibition was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry after siRNA treatment. The results showed that the cell growth of siRNA treated group was inhibited, and the apoptosis of NB4 could be induced. The siRNA targeting PML-RARα mRNA might be a valid therapy of acute promyelocytic leukemia.
Objective To establish hepatocellular carcinoma (HCC) cell lines which olig-expressed IGF1R gene stably． Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents． After transferred, cells were selected with G418 to obtain positive clones. The expressions of IGF1R, cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot． Cell growth curve were painted. Results Two cell lines clones were screened olig-expressing IGF1R gene stably． The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased (P＜0.05). Conclusion The HCC cell lines for olig-expressing IGF1R gene stably are established successfully．The plasmid pSUPER-IGF1R-siRNA can inhibit the growth of SMMC7721 and Hep3B cell lines, and the expression of cyclin D1.