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<p><b>OBJECTIVE</b>To construct a lentiviral vector carrying the γ-synuclein(SNCG) gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and then investigate the inhibition of the growth and invasion capacity of SW1116 cells.</p><p><b>METHODS</b>RNA interference fragment was designed according to the SNCG sequence (GenBank: No.NM003087.2), and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP (RNAi group) and LV-SNCG-NC-EGFP (NC group) were constructed and packaged to produce lentivirus venom. The supernatants of different virus-producing cells were used to transfect SW1116 cells respectively. Wild SW1116 cells were used as blank control (CON group) EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively.</p><p><b>RESULTS</b>Recombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirus could effectively infect SW1116 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV-SNCG-NC-EGFP was 8×10(8) TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression (1.009±0.161 vs. 0.114±0.030, P=0.009), and showed tremendous silencing effect as 76.8%(P<0.05). SNCG protein expression was also significantly reduced (RNAi:12.001±2.884, NC:32.443±4.731, CON:34.308±6.920, P<0.05). After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively(P=0.036). In soft agar assay, clones in RNAi group were smaller[RNAi:(0.582±0.103) mm, NC:(1.863±0.316) mm, CON:(1.749±0.525) mm]. Colony formation rate of RNAi group was down to (17.1±3.5)%, which was significantly lower than (36.5±4.3)% in NC group and (33.8±3.9)% in CON group. In migration test, the number of invasion cell was 37.4±9.3 in RNAi group, which was significantly less than 112.3±8.6 in NC group and 100±0.0 in CON group.</p><p><b>CONCLUSION</b>Expression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.</p>
Assuntos
Humanos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais , Patologia , Vetores Genéticos , Lentivirus , Interferência de RNA , RNA Mensageiro , RNA Interferente Pequeno , Genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , gama-Sinucleína , GenéticaRESUMO
@#The purpose of this research was to study the effect of Xingxiong sodium chloride injection on cerebral ischemia-reperfusion injury in rats. Rats were divided into five groups, namely sham group, model group, ginkgolide injection group(3 mg/kg), Xingxiong sodium chloride injection high-dose group(14 mg/kg)and low dose group(7 mg/kg). Except for the sham group, animals in other groups were subjected to ischemia for 2h and reperfusion for 72 h by middle cerebral artery occlusion(MCAO)with thread technique, and then drugs were administered continuously by tail intravenous injection during reperfusion period for 3 days. The neurological deficit score and the histopathological score were evaluated; infarction ratio, brain water content and biochemical indexes of animals in each group were determined. Compared with model group, reduction of neurological deficit score, brain water content and infarction area were observed obviously at 7 mg/kg and 14 mg/kg of Xingxiong sodium chloride injection. Additionally, reduction of histopathological score, H2O2 and MDA content, increase of the level of GSH, GSH-Px, SOD and also the capacity of inhibition of superoxide anion radical(·O-2)and hydroxyl radial(·OH)were observed at 14 mg/kg. The results suggested that Xingxiong sodium chloride injection could effectively enhance the ability of anti-oxidation in the brain tissues, and protect brain from ischemia-reperfusion injury.
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<p><b>OBJECTIVE</b>To construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.</p><p><b>METHODS</b>Total RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.</p><p><b>RESULTS</b>Human γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).</p><p><b>CONCLUSION</b>Expression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.</p>