| Objective:When mesenchymal stem cells was cultured and induced to adipocyte in vitro, the cell morphology had a substantial change from the initial fusiform to round and ultimately generation a mature adipocytes. By adjusting its protein kinase (ROCK) and its downstream protein MYPT, MLC, etc, Rho can regulate actin polymerization of actin cytoskeleton, thereby affecting cell polarity, morphology and adipogenic situation. RhoAG14V is from the 14th glycine mutated to valine,and can continutively activated to express. Therefore, we will construct RhoAG14V stable cell lines to be used for adipogenic differentiation, and explore the effect of overexpression of RhoAG14V for adipogenic differentiation, where and how it is to play the role of Rho A/ROCK signaling pathways. We expected that it could provide a new theoretical reference for the effective regulation of poultry fat deposition and treatment or prevention of human obesity-related diseases.Methods:1、Construction the C3H10T1/2 cell with overexpression of Rho AThe PlatE cells were seeded in 80% of 6 cm dish,24 hours later, pMSCV-puro-HA-DD-hRhoA-DA were transfected into PlatE cells using Sage Creation according to the introduction. After 8 hours, replace the fresh DMEM medium.Then collect the virus 48h and filter by 0.45μm membrane, standby placed in -80℃. C3H10T1/2 cells were seeded at 50% -60%,24 hours later, replace the fresh DMEM mediumand add 2μL polybrene to make the final concentration of 10 μg/mL, is placed 37℃ for 30 min.At the same time the virus was thawed on ice, then the virus was added and cubated. Another 48 hours later, the DMEM containing puromycin (10μg/mL) was used to screening for resistance to two weeks.Then the cell was cultured with DMEM medium containing puromycin(5μg/mL) to construct cell lines with RhoAG14V over-expression cells.Verified by sequencing RT-qPCR and Western blot methods.2、Effect of overexpression of RhoAG14V gene on triglycerides accumulation of C3H10T1/2 cells after adipogenic differentiationRhoAG14V and C3H10T1/2 groups were were plated at concentration of 5×104/cm2. After two days of the cells reached confluency and started the adipogenic differentiation experiments. C3H10T1/2 group were cultured in medium A that complete medium containing containing 1.0 μmol/L dexamethasone,0.5 mmol/L IB MX,10 mg/L insulin,5.0μmol/L rosiglitazone for 2 days.Then the cells were cultured in medium B that complete medium containing containing 1.0 μmol/L dexamethasone and 10 mg/L insulin for 2 days. The next days cells replace the medium alternative medium A and B every 2 days until the end of the induction process. RhoAG14V group on the basis of C3H10T1/2 groups at day 0 added added shield 1(1μm/mL) protecting agent,the other dealing with to the same group adipogenic differentiation 8d end of the experiment. Stained oil red O was extracted with isopropyl alcohol and quantified by measuring the optical absorbance at 490nm.3、Effect of overexpression of RhoAG14V gene on relative gene expression of C3H10T1/2 cells during adipogenic differentiationTotal RNA was prepared using by TRIzol reagent,β-actin as an internal reference, using real-time quantitative RT-qPCR detection of gene PPARy, C/EBPa and RhoA expression.4、Effect of overexpression of RhoAG14V gene on relative protein expression of C3H10T1/2 cells during adipogenic differentiationWestern blot detection RhoA, ROCK, MYPT1,p-MYPT, MLC, p-MLC and protein expression of adipogenic genes PPARy.5、Effect of overexpression of RhoAG14V gene on changing of skeleton C3H10T1/ 2 cells during adipogenic differentiationIn order to observe the distribution of cellular actin cytoskeleton, the cells were induced Od, 1d,3d,5d,7d, respectively。Then using Anti-stainTM488 Fluorescent Phalloidin and DAPI stain the cells. The cell climbing piece were washed two times with PBS and fixed with 4% paraformaldehyde for 15 min,then with Anti-stainTM488 Fluorescent Phalloidin dilution of 1:100 stained cell actin cytoskeleton。That were washed to remove Anti-stainTM488 Fluorescent Phalloidin antibody with PBS, stained nucleus with DAPI.Using 1% BSA-PBS mounted and observed the changing of distribution of actin cytoskeleton in the confocal microscope.Result:1-. Construction the C3H10T1/2 cell with overexpression of RhoAConfiremed By sequencing、RT-qPCR and Western blot。Sequence analysis showed that stable cell lines overexpressing RhoAG14V gene sequence alignment with human sequence databases (GenBank), which RhoA gene glycine at position 14 was changed to valine, the rest of the sequence are exactly the same. Compared with the control group, RT-qPCR analysis showed C3H10T1/2 the Gene expression level of stably overexpressing cell lines expressing RhoAG14V gene is 33 times. Western blot analysis showed C3H10T1/2 the protein expression level of stably overexpressing cell lines expressing RhoAG14V gene is 1.23 times (p<0.01).2、Effect of overexpression of RhoAG14V gene on triglycerides accumulation of C3H10T1/2 cells after adipogenic differentiationwhen mesenchymal stem cells C3H10T1/2 cells were induced differentiation to 8d, the cells present the typical adipocyte phenotype,which more than 95% of the cells are rich in large lipid droplets; 65% of the RhoAG14V cells are rich in lipid droplets, and lipid droplets smaller. Oil red O staining significantly less fat than a set of colors, which is consistent with the value of OD 490 nm of the isopropyl alcohol extracted intracellular oli red Ostaining of lipid droplets.3> Effect of overexpression of RhoAG14V gene on relative gene expression of C3H10T1/2 cells during adipogenic differentiationReal-time quantitative RT-qPCR results showed that in the process of induction of differentiation the overexpression of RhoA gene group present a gradual downward trend, and it is higher than the C3H10T1/2 the control group during adipogenic differentiation. But C/EBPa and PPARy expression levels in the overexpression of RhoAG14V were lower than the control group C3H10T1/2 cells during adipogenic differentiation.4-, Effect of overexpression of RhoAG14V gene on relative protein expression of C3H10T1/2 cells during adipogenic differentiationWestern blot detected RhoA, ROCK, change MYPT1, p-MYPT1, MLC, p-MLC and PPARr expression between RhoAG14V group and C3H10T1/2 cells during the induced adipogenic differentiation.(1) In the induced differentiation process, RhoA protein expression showed a gradual decreasing trend both RhoAG14V group and C3H10T1/2group,but in the whole process of induction of differentiation expression level of RhoAG14V protein is always higher than C3H10T1/2 group.(2) In the induced differentiation process, ROCK, p-MYPT1,P-MLC proteins expression levels of RhoA14V group maintained a steady state; C3H10T1/2 group increased and decreased after a period of time. Excepted the third days, in the RhoAG14V group,the expression levels of ROCK, p-MYPT1, P-MLC are higher than C3H10T1/2 groups(3) During adipogenic differentiation,the expression amount of PPARy of RhoAG14V group are always lower than the C3H10T1/2 group, and showed a gradual downward trend. Expression level of PPARy protein increased gradually in the C3H10T1/2 group.5、Effect of RhoAG14V gene on changing of skeleton C3H10T1/2 cells induced into fat cellsIn the confocal microscope,C3H10T1/2 cells were observed to spindle, filamentous actin green fluorescence.Ordered reticular actin surrounded cell nuclear and formateed stress fiber in occasional. RhoAG14V cells actin obviously showed concentric retraction, Some filopodia and lamellipodia surrounded the cell nuclear. with the induction time was gone, the cell cytoskeleton groups were gradually relaxed.Conclusion:Overexpression of RhoAG14V gene can reduce the expression amount of PPARy protein that is one of the adipogenic differentiation marker protein by regulating ROCK, MYPT and p-MYPT, MLC and p-MLC protein that relatived with cytoskeletal contraction in the RhoA/ROCK signaling pathway; As the same time overexpression of RhoAG14V gene can reduce the expression level of C/EBPa and PPARy that be associated adipogenic differentiation from the transcriptional level. Finally the adipocytes ability of C3H10T1/2 cells was decreased. |
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