| Background: Osteonecrosis is a very common joint disease in clinical. The causes of this diease are very complex, and the morbidity rate is high. The patients who suffer this disease might need surgery treatment once the symptom occurred, thus this disease causes a great threat on life quality and health of patients. Nowadays, non-traumatic Osteonecrosis of femoral head(ONFH) become more and more common, amount of research data has been revealed that Chronic femoral head necrosis associated with hormone and alcohol intake. In China, chronic alcohol poisoning is one of the leading causes of ONFH. In rats, mice, rabbits and chickens, animal will appear evident phenomenon of femoral head necrosis after long-term use of high doses of alcohol lavage treatment for a certain period of time. Bone marrow stromal cells(BMSCs) are a kind of pluripotent stem cells which mainly to differentiate into osteoblasts, and can differentiate into fat cells, myocardial cells andnerve cells, etc. The BMSCs cultured in vitro can not only differentiate into fat tissues and also loss osteogenesis ability after high dose alcohol treatment.Peroxisome proliferators-activated receptor γ(PPARγ) is a key transcription factor of adipogenesis, it is highly expressed in adipocytes and the activation of PPARγ is closely associated with cell differentiation fate. A large number of studies have shown that the activation of PPARγ occurred in BMSCs will block cell osteogenic differentiation and reverse the cell differentiation fate to adipogenesis, this process known as loss of bone mass and disturb bone or joint repair ability in the development of bone osteonecrosis.MicroRNAs are short chain non-coding RNA of about 20 to 24 nucleotides length. According to its specific nucleotide sequence, different types of micro RNAs can target to different gene’s m RNA, thus micro RNAs can regulate the gene expression in m RNA post-transcription level. The regular expression status of micro RNAs is very important to maintain normal life activities, and about 60% human genes are under the regulation of micro RNAs. One micro RNA can targeted to multiple genes, and one gene can also be targeted by different micro RNAs. Through biological information database, we found that micro RNA-27 a can inhibit PPARγ gene expression through target to PPARγ gene m RNA by pairing their complementary sequence, and this process can initial a recovery ability on osteogenesis of BNSC in order to prevent and inhibit ONFH.Objective: This study aim to investigate the preventive ability of micro RNA on ONFH and the function of micro RNA-27 a on targeting PPARγ in order to regulate its expression level, meanwhile, this project also put a discussion on the influence of alcohol on adipogenic and osteogenic differentiation of BMSC in vitro via micro RNA-27 a mimics transfection.Methods: 1. Grouping: The rat BMSCs were cultivated in a concentration of 1×106/cm2 in the six-wells plate, and conduct electroporation method for micro RNA mimicstransfection when the cell cluster reach approximate 80%, and randomly grouping each group as follow: ① Control: normal cultivated BMSCs, no special treatment. ② Model: add 0.09 mol/L ethanol into medium as induction reagent. ③ Micro RNA-27a: the BMSCs were transfected with micro RNA-27 a mimics and add 0.09 mol/L ethanol into medium as induction reagent at the same time. ④ NC: the BMSCs were transfected with negative control mimics(random and irrelevant sequence) and add 0.09 mol/L ethanol into medium as induction reagent at the same time2. The match condition between micro RNA-27 a and PPARγ 3’-UTR were detected via luciferase reporter experiment.3. Real-time Quantitative PCR(q PCR) was used for investigate the PPARγ and Runx2 m RNA expression differences on each group after alcohol induction treatment.4. Alkaline phosphatase(ALP), Type I Collagen and osteocalcin(OCN) protein level of BMSCs on each group after alcohol induction treatment were detected via enzyme-linked immune-sorbent assay(Elisa).5. The changes of PPARγ and Runx2 protein level of each group after alcohol induction treatment were determined by western blot assay.6. Oil red staining method was conducted to investigate the adipogenesis and adipogenic degree of each group BMSCs after alcohol induction treatment.7. Enzymatic colorimetric method kit were used for detecting the differences of TG content in each experimental group.Results: 1) Luciferase reporter experiment has been authenticate that, the match between micro RNA-27 a and PPARγ 3’-UTR was solid, therefore, PPARγ is a target gene of micro RNA-27a’s.2) The results of qPCR indicate that, the PPARγ expression of model group and NC group were higher than control group and micro RNA-27 a group afteralcohol-induction treatment(P<0.01), but the differences between model group and NC group, plus control group and micro RNA-27 a group have no significance(P > 0.05); To the contrary, the Runx2 expression of control group and micro RNA-27 a group were higher than NC group and model group(P<0.01), but difference between NC group and model group, plus control group and micro RNA-27 a group were insignificant(P>0.05).3) ALP detection results indicate that, compare wtih control group, the ALP protein level of model group and NC group were decreased after alcohol-induction treatment(P<0.01), and micro RNA-27 a group status was similar to control group, and the difference between these two groups has no significance(P>0.05).4) Type I Collagen determination results indicate that, compare wtih control group, the Type I Collagen protein level of model group and NC group were decreased after alcohol-induction treatment(P<0.01), meanwhile, the micro RNA-27 a group status was similar to control group, and the difference between these two groups has no significance(P>0.05)5) OCN determination results indicate that, the OCN protein level of model group and NC group were decreased comparing wtih control group after alcohol-induction treatment(P<0.01), and the difference between micro RNA-27 a group and control group has no significance(P>0.05).6) Western blot results showing that, after alcohol-induction treatment, the PPARγ expression level of micro RNA-27 a group and NC group were increased dramatically comparing with control group(P<0.01), and the expression level of micro RNA-27 a group and control group were very similar(P>0.05); Meanwhile, the Runx2 expression level showing a reverse change trend. The Runx2 expression level of model group and NC group and were decreased comparing with control group(P<0.01), but the difference between micro RNA-27 a group and control group has no statistical significance(P>0.05).7) Oil red O staining results indicate that, there are rarely no lipid droplet production in control group; The model group and NC group have huge amount of lipid droplets, showing brownish red color after react with oil red O molecule, in the cytoplasm; the number of adipocytes of micro RNA-27 a group and control groupwere less than model group and NC group(P<0.01). but the difference between micro RNA-27 a group and control group has no statistical significance(P>0.05).8) The results of TG content detection say that, the TG content of model group and NC group has a higher expression level than control group and micro RNA-27 a group(P<0.01). And there is no statistical significance between micro RNA-27 a group and control group(P>0.05).Conclusions: 1. micro RNA-27 a has the ability to target PPARγ and down-regulate its expression level, and micro RNA-27 a also can decrease adipocytes production rate, lower the lipid droplets output and TG content, thus inhibit alcohol-induced adipogenic differentiation of BMSCs.2. Micro RNA-27 a can increase the expression level of Runx2 and rise ALP, Type I collagen and OCN content in BMSCs during alcohol induction treatment, and promote the osteogenic differentiation ability of BMSCs.3. Micro RNA-27 a is capable of preventing the initiation and development of ONFH. |
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