| BackgroundAlcohol-induced osteonecrosis of the femoral head(ONFH) is a common orthopaedic clinical disease and the incidence is increasing year by year. This disease is often seen in young patients, with bilateral occurred. The patients suffering from ONFH will progress to a collapse of the femoral head, resulting in loss of joint function if they do not receive appropriate treatment, which seriously affect the patient’s quality of life. Most of the patients with late stage ONFH will eventually require a total hip replacement(THA) and the revision hip arthroplasty will inescapable, which is not ideal, especially in younger patients.Studies show that alcohol abuse can stimulate the elevation of peroxisome proliferator-activated receptor-γ(PPARg) gene expression in bone marrow stem cells, which promote adipogenic differentiation, inhibit the osteogenic differentiation of these cells. The accumulation of fatty tissue in the femoral head can block blood circulation, and lead to high intra-osseous pressure, which finally result in ONFH.Down-regulation of PPARg expression may inhibit adipogenic differentiation and maintain the osteogenic differentiation, which can decrease the incidence of ONFH. Calcitonin gene-related peptide(CGRP) is a neuropeptide gene which is related to bone growth and metabolism. CGRP can promote the multiplication capacity and osteogenic activity of bone marrow stem cells, which play an important role in the regulation of bone formation and resorption. Therefore, combined regulation of the expression of PPARγ and CGRP gene can inhibit adipogenic differentiation and enhance the osteogenic differentiation of bone marrow stem cells, which enlighten a novel approach for the prevention of ONFH.ObjectiveThe main purpose of this study was to construct the recombinant vector carrying a small interfering RNA targeting PPARg gene and the CGRP gene, to observe the effect of combined regulation of the expression of PPARγ and CGRP gene on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs). Then, BMSCs which transfected with the recombinant vector transplanted into the femoral head of rabbit model of ONFH, to observe the preventive effect of alcohol-induced ONFH, which provide a novel experiment condition for the prevention or treatment of ONFH. This study is divided into the following three parts:PartⅠConstruction and identification of the recombinant vector silencing PPARγ and expressing CGRP geneMethods(1)The PPARγ si RNA oligonucleotides were designed according to the gene sequence of PPARγ in Gen Bank, Bam HⅠand Hind Ⅲ restriction enzyme digestion residues were introduced at 3′-end and 5′-end of these oligos and one base of the Bam HⅠrestriction enzyme digestion site was changed when we designed.(2) Designed and amplified rabbit CGRP gene open reading frame(ORF) clone with Mlu I restriction enzyme digestion residues. The CGRP ORF was linked with TGFP fusion protein by flexible peptide with the sequence GGT GGC GGT GGA TCC GGT GGC GGT GGC TCC.(3) After annealing, the double-stranded hairpin c DNA was ligated into the p GFP-V-RS vector to obtain the silencing vector p GFP-V-RS-si PPARγ, which can down-regulate expression of the PPARg gene. The ORF c DNA of CGRP was also ligated into the p GFP-V-RS vector to obtain the expression vector p GFP-V-RS-ex CGRP, which expresses the CGRP gene. Meanwhile the ORF c DNA of CGRP was cloned into the multiple cloning site(MCS) downstream of the CMV promoter to obtain the double-gene vector p GFP-V-RS-si PPARγ-ex CGRP, which can down-regulate expression of the PPARg gene and express the CGRP gene.ResultsWe successfully constructed three recombinant vectors after enzyme identification and DNA sequencing, which provide the experiment condition for observe the combined regulate effect of the recombinant vectors on alcohol induced adipogenic and osteogenic differentiation of BMSCs.Part The eⅡ ffect of combined regulation of the expression of PPARγ and CGRP on alcohol-induced adipogenic differentiation of BMSCsMethods(1) Autologous primary BMSCs were harvested using lymphocyte separation medium by density gradient centrifugation, and the surface markers of CD29, CD34, CD44, CD45, or CD105 were analysed by flow cytometry.(2) The recombinant vectors were transfeced into BMSCs by BTX ECM 2001 system and alcohol at 0.09mol/L was added to the culture medium of all groups requiring alcohol induction. MTT proliferation assay was used to determine the cell proliferation. The expression of PPARγ, CGRP, Runx2 and osteocalcin m RNA and protein were determined by Taqman RT-PCR and Western blotting. The content of triglyceride(TG) in the cells, the activity of alkaline phosphatase(ALP), the content of laminin, collagen type I and osteocalcin in the medium were determined by enzyme-linked immunosorbent assay(ELISA). The alkaline phosphatase(ALP) staining and red oil “O” staining were also taken.Results(1)The results of cell identification by flow cytometry revealed that BMSCs were positive for CD29, CD44, and CD105 with positive rates of 99.86%, 99.34%, and 99.65% respectively. The cells were negative for CD34 and CD45, with positive rates of 1.27% and1.45%.(2)In the group of BMSCs which transfected with the silencing vector p GFP-V-RS-si PPARγ showed that, the cells’ proliferation was slightly decreased than that in the normal group and there is no significantly difference when two groups were compared(P>0.05). There was no significant difference when compare with normal group regarding to the expression of PPARγ, Runx2 and osteocalcin in both m RNA and protein levels(P>0.05). TG content, ALP activity, content of laminin, collagen type I and osteocalcin in the medium were no significant difference when compare to that in the normal group(P>0.05). At day 14, the results of ALP staining showed that the cells staining for blue/purple partly. At day 21, no fat droplets in cells were found or rarely found, and Oil red O staining assay showed that no or few cells stained positive, which is similar to that in the normal group.(3) In the group of BMSCs which transfected with the expression vector p GFP-V-RS-ex CGRP showed that, cells’ proliferation was slightly lower than that in the normal group and there is no significantly difference(P>0.05). CGRP m RNA and protein were expressed stably. Expression of PPARγ m RNA, protein and TG content were all significantly higher than that in the normal group(P<0.05), while Runx2, osteocalcin m RNA and protein, ALP activity, the content of laminin, collagen type I and osteocalcin in the medium were similar to that in the normal group(P>0.05). At day 14, the results of ALP staining showed that the cells staining for blue/purple partly, which is similar to that in the normal group. At day 21, fat droplets which like annular black particles with strong refraction were found in cells, and Oil red O staining assay showed that contained reddish-orange fat droplets in their cytoplasm.(4) In the group of BMSCs transfected with the double-gene recombinant vector p GFP-V-RS-si PPARγ-ex CGRP showed that, cells’ proliferation was almost the same as that in the normal group(P>0.05), and CGRP m RNA and protein were stably expressed. Expression of PPARγ m RNA, protein, and TG content were similar to the normal group, there is no significant difference(P>0.05). The levels of Runx2, osteocalcin m RNA and protein, ALP activity, the content of laminin, collagen type I and osteocalcin in the medium were all significantly higher than that in the normal group(P<0.05). At day 14, the results of ALP staining showed that a large number of cells staining for blue/purple, which is more than that in the normal group. At day 21, no fat droplets in cells were found or rarely found, and Oil red O staining assay showed that no or few cells stained positive, which is similar to that in the normal group.Part Ⅲ The preventive effect of combined regulation of the expression of PPARγ and CGRP on alcohol-induced ONFH in RabbitsMethods(1) Take 80 healthy New Zealand rabbits, and divided into 5 groups randomly. Horse serum(10ml/kg) was injected through auricular vein twice with the interval of 3 weeks for sensitive condition. Animals were administerd with spirits(the volume fraction of alcohol was 46%) 2 week later. 25μl of BMSCs transfected with recombinant vectors was injected into one of femoral head randomly on the 1st day in the 1st, 3rd and 6th week. The animals in the experiment were sacrificed in batches on 4 and 8 weeks periodically.(2)Experiment grouping: Group S: rabbits were treated with alcohol and recombinant vector p GFP-V-RS-si PPARγ-ex CGRP after sensitized by horse serum. Group M: rabbits were treated with alcohol after sensitized by horse serum. Group Con: rabbits were treated with alcohol and empty vector p GFP-V-RS after sensitized by horse serum. Group H: rabbits were sensitized by horse serum only. Group N: rabbits with no treatment served as controls.(3)Femoral head specimens were cut into two symmetrical parts along a coronal plane. One half of the specimen was stained with HE and the other was stain with SudanⅢ. The following parameters were assessed: 1. the incidence of osteonecrosis;2. fraction of trabecular bone area; 3. percentage of empty osteocyte lacunae; 4. the average diameter of the largest adipocyte.(4)The expression of the PPARγ, CGRP, Runx2, osteocalcin m RNA and protein were determined through Taq Man real time-PCR and Western blotting.Results(1) 8 weeks after treatment, gross examination revealed that the femoral heads of rabbits in the group M and group Con became whitish, soft, and easily cut. Under microscopy, we found evidence of bone marrow necrosis, diminished hematopoiesis, increased fat, thinning of trabecular bone, with evidence of trabecular breakage. However, no such pathologic change was observed in Group N, Group H and Group S.(2)4 week after treatment, the percentage of empty osteocyte lacunae was increased in animals of group M and group Con, and was significantly higher compared with group N(P<0.05),the percentage of empty osteocyte lacunae in group H and group S was similar to group N(P>0.05). 8 weeks after treatment, the average diameter of the largest adipocyte, the percentage of empty osteocyte lacunae were increased in both the group M and group Con, compared with the animals in group N(P<0.05), and the fractional area of trabecular bone was decreased compared with group N(P<0.05). No differences the average diameter of the largest adipocyte, the percentage of empty osteocyte lacunae and the fractional area of trabecular bone were seen in group S and group H, compared with the animals in group N(P>0.05).(3)At week 4 and 8, the expression level of PPARγ m RNA and protein in the group M and group Con was significantly higher than that in the group N(P<0.05), the expression of CGRP, Runx2 and osteocalcin m RNA and protein were lower than that in the group N(P<0.05). The expression level of PPARγ m RNA and protein in group S was similar to the group N(P>0.05), the expression of CGRP, Runx2 and osteocalcin m RNA and protein were higher than that in the group N(P<0.05). The expression level of PPARγ, CGRP, Runx2 and osteocalcin m RNA and protein in group H was similar to the group N(P>0.05).Conclusions(1) Three recombinant vectors were successfully constructed: the silencing vector p GFP-V-RS-si PPARγ, which can down-regulate expression of the PPAR? gene; the expression vector p GFP-V-RS-ex CGRP, which expresses the CGRP gene; the double-gene vector p GFP-V-RS-si PPARγ-ex CGRP, which can down-regulate expression of the PPAR? gene and express the CGRP gene.(2) The experiment in vitro confirmed that, the double-gene vector p GFP-V-RS-si PPARγ-ex CGRP can efficiently block PPARγ gene expression of alcohol induced BMSCs, and promote the CGRP gene expression, which suppressed adipogenic differentiation and improved osteogenic potential of the cells.(3)The experiment in vivo confirmed that, combined regulation of PPARγ and CGRP genes can significantly reduce the fat deposition in the femoral head of rabbit induced by alcohol, and enhance the osteogenic differentiation of BMSCs, promote the repairation and reconstruction of bone tissue, which can efficiently prevent alcohol-induced ONFH of rabbits.(4) In this study, we used double-gene modified BMSCs, which can block PPARγ gene expression and inhibite adipogenic adipogenic differentiation, meanwhile promote CGRP gene expression and enhanced osteogenic differentiation. This innovative technology may provide a novel approach for the prevention of ONFH. |
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