| Purpose:This experiment from the TCM theory of "kidney advocate bone marrow" as a starting point,choose the typical "nourishing kidney Yin" left to pill and "warming Yang" right to pellets,and will be left to pills,pills right to disassemble,on both sides of the communist party of China some medicine for a common group,to the left to bolus of nourishing kidney Yin drugs for ziyin medicine group,with right belongs to the pill of warming Yang to fill to Yang medicine group,positive control group for estradiol ketone pills,to ovarian rats(postmenopausal osteoprosis,PMOP)of bone marrow mesenchymal stem cells(ipads mesenchymal stem cells,BMSCs)as the research object,is on the left and right to pill and its demolition party to ovary differentiation mechanism of rat BMSCs into fat,to explore on the left and right to pill and its demolition party to ovary differentiation of rat BMSCs into fat possible mechanism,and to the left and right to provide experimental basis to pill in the treatment of osteoporosis.Material and methods: Bilateral ovaries method is adopted to establish the model of postmenopausal osteoporosis,SPF 2 months 90 SD female rats childless,weight range of 200~ 220 g,adaptive feed after 1 week according to the digital random method were randomly divided into normal group,the control group only ten only 70 only,and the surgical control only near the ovaries of adipose tissue,scores of bilateral ovaries enucleation.Survived after the operation,the rats again stratified random divided into 7 groups by weight,so the grouping results are as follows: normal group(CON)10,10 only control(SHAM),model group(OVX)8,left to pill group(ZGW)8,right to pill group(YGW)8,common prescription(GTY)8 ziyin medicine group(ZYY),and ultimately only medicine group(BYY)8 fill jiale group(BJL),only.Drug dose per kg of body weight in rats of dosage is 6.3 times of calculation,and each time to rats by gastric quantity is 2 times of the normal dose,daily,1times a filling and CON,OVX and SHAM groups distilled water;the rest groups respectively take corresponding preparation good Fried liquid water.Continuous dosing after 12 weeks in ultra clean machine to extract the above each rat BMSCs P0,determined by MTT method isused to detect BMSCs proliferation rate,oil red O staining method to detect the formation of lipid droplets,and the policy of real-time PCR method to detect the peroxidase body growth activated receptor ?(peroxisome proliferator-activated receptor ?,PPAR ?),lipoprotein lipase(lipoprotein lipase,LPL),fatty acid binding protein 4(fatty acid binding protein 4,FABP4),CCAAT/enhancer binding protein-a(CCAAT enhancer binding protein a,C/EBPa),CCAAT/enhancer binding protein-?(CCAAT enhancer binding protein ?,C/EBP ?)m RNA expression.Using Western blot method to detect PPAR ?,LPL,FABP4,C/EBPa,C/EBP ?protein expression.Results:1Identification of BMSCsBy the detection of flow cytometry,the expression rate of CD29 of BMSCs is 98.1%,of CD90 is 95.9%,of CD11 is 17.1% and of CD45 is 36.0%.2Effects of Zuogui Pill,Yougui Pill and their Prescriptions on the Proliferation of BMSCs of the PMOP Rats.2.1The Drawing of Growth Curve of BMSCsAfter the cells are inoculated,the incubation period is approximately from 1 to 2 days.From the third day,the cells begin to grow and enter the logarithmic growth phase.The peak of cell growth is on the 5thto 6thday,and finally reaches the platform stage.It is found that BMSCs grows in S shape.2.2Effects of Zuogui Pill,Yougui Pill and their Prescriptions on the Proliferation of BMSCs of PMOP Rats.The results show that the effects of Zuogui Pill,Yougui Pill and their prescriptions on the proliferation of BMSCs of PMOP rats are time-dependent.On the first day,compared with CON group,ZGW and GTY group have significant statistical differences on the effects on the proliferation(P<0.05).However,compared with that of OVX group,there are no significant statistical differences existing in the other groups(P>0.05).On the third day,compared with CON group,ZGW?YGW?ZYY?BYY?BJL grouphave significant statistical differences(P<0.05);compared with OVX group,only BJL group has significant statistical differences(P<0.05);and compared with ZGW group,only BJL group has significant statistical differences(P<0.05).On the 5th day,compared with CON group,that of ZGW?YGW?GTY?ZYY?BYY and BJL group have significant statistical differences(P <0.05);compared with OVX group,ZGW,YGW and BJL group have significant statistical differences(P <0.05),compared with ZGW group,GTY and ZYY group have significant statistical differences(P <0.05).On the 7thday,compared with CON group,OVX,ZGW,YGW,GTY and BJL group have significant statistical differences(P <0.05);compared with OVX group,ZGW,YGW,GTY and BJL grouphave significant statistical differences(P<0.05);compared with ZGW group,SHAM,YGW,GTY,ZYY,BYY and BJL group arestatistically significant(P<0.05).3On the left and right to pill and its demolition party PMOP rats induction and differentiation of BMSCs into fat3.1left and right belongs to the pill and its open side of BMSCs lipid drops expression of PMOP ratsOil red O staining method to detect PMOP rats BMSCs expression of lipid droplets.A concomitant induction after 10 days,BMSCs was observed microscopically by shuttle deformation for the oval or round,uneven size appeared in the cytoplasm of a lipid drops.After oil red O staining,lipid droplets present a bright red.Compared with CON group,the rest of the group number of lipid droplets increases;Compared with OVX group,the rest of the group number of lipid droplets decrease;Compared with BJL group,ZGW group,YGW group,the GTY group,ZYY medicine group and BYY lipid drops reduced,compared with the ZGW group,SHAM group,the YGW group fat drips to reduce,GTY group,ZYY and BYY lipid drops by the increase in the number.3.2Effects of Zuogui Pill,Yougui Pill and Their Prescriptions onm RNA and Protein Expressions of Cs PPAR? in BMSCs of POMP Rats.Compared with that of CON group,the m RNA expression of PPAR? of BMSCs in SHAM and BJL group are not significantly statistical different(P < 0.05);the m RNA expressions of PPAR? of BMSCs in OVX,ZGW,YGW,GTY,ZYY and BYY group can be significantly raised(P<0.05).Compared with that of OVX group,the m RNA expressions of PPAR? of BMSCs in SHAM,ZGW,GTY and BJL group can be significantly reduced(P<0.05).Compared with that of ZGW group,the m RNA expressions of PPAR? of BMSCs in YGW and BYY group increase significantly(P<0.05),and that in BYY group decreases significantly(P<0.05).Compared with that of CON group,the protein expressions of PPAR? of BMSCs in SHAM,ZGW,BYY and BJL group are not significantly different(P>0.05);the protein expressions of PPAR? of BMSCs in OVX,YGW,GTY and ZYY group BMSCs increase significantly(P<0.05).Compared with that of OVX group,the protein expressions of PPAR?of BMSCs in SHAM,ZGW,GTY,BYY and BJL group can be reduced significantly(P<0.05),that of YGW group can be raised significantly(P<0.05),that of ZYY group is not significantly statistical different(P>0.05).Compared with that of ZGW group,the protein expressions of PPAR? of BMSCs in YGW and ZYY group increase significantly(P<0.05),that in SHAM group decreases significantly(P<0.05).3.3Effects of Zuogui Pill,Yougui Pill and Their Prescriptions on m RNA and Protein Expressions of LPL in BMSCs of POMP Rats.Compared with CON group,SHAM,YGW and BJL group do not have significantly statistical differences on m RNA expressions of LPL(P>0.05);m RNA expressions of LPL in the other groups increase significantly,and the differences have statistical significance(P<0.05).Compared with that of OVX group,in SHAM,ZGW,YGW,GTY,ZYY and BJL group,m RNA expressions of LPL in BMSCs can be decreased significantly(P<0.05).Compared with that of ZGW group,m RNA expression of LPL in YGW decreases significantly(P<0.05),but those in ZYY and BYY group have varying degrees of increase(P<0.05).Compared with CON group,SHAM,ZGW,YGW and BJL group do not have significantly statistical differences on protein expression of LPL in BMSCs(P>0.05).Protein expressions of LPL in the OVX,GTY,ZYY and BYY groupincrease significantly(P<0.05).Compared with that of OVX group,protein expressions of LPL in BMSCs in SHAM,ZGW,YGW and BJL group can be decreased significantly(P<0.05);and there are no significantly statistical differences in GTY,ZYY and BYY group(P<0.05).Compared with that of ZGW group,protein expressions of LPL can be decreased significantly in SHAM and YGW group,and the statistical differences are significant(P < 0.05).There are notsignificantly statistical differences in other groups(P>0.05).3.4Effects of Zuogui Pill,Yougui Pill and Their Prescriptions on m RNA and Protein Expressions of FABP4 in BMSCs of POMP Rats.The results show that,compared with CON group,SHAM and BJL group do not have significantly statistical differences on m RNA expressions of FABP4(P >0.05);m RNA expressions of FABP4 in BMSCs of OVX,ZGW,YGW,GTY,ZYY and BYY group increase significantly(P<0.05).Compared with OVX group,YGW and ZYY group do not have significantly statistical differences on m RNA expression of FABP4(P>0.05),in SHAM,ZGW,GTY and BJL group,m RNA expression of FABP4 in the BMSCs can be decreased significantly(P<0.05).Compared with that of ZGW group,m RNA expressions of FABP4 in SHAM and BJL group can be decreased significantly(P<0.05),and those in YGW,ZYY and BYY group are more than the normal(P<0.05).Compared with CON group,SHAM,ZGW,GTY and BJL group do not have significantly statistical differences on protein expressions of FABP4(P>0.05),in OVX,YGW,ZYY and BYY group,proteinexpressions of FABP4 in the BMSCs increase significantly(P<0.05).Compared with that in OVX group,in SHAM,ZGW,GTY and BJL group,proteinexpressions of FABP4 in the BMSCs can be decreased significantly(P<0.05),ZYY and BYY group do not have significantly statistical differences on protein expressions of FABP4.Compared with ZGW group,YGW and GTY group do not have significantly statistical differences on protein expressions of FABP4(P>0.05),that in BJL group can be decreased significantly(P<0.05),and those both in ZYY and BYY group can be increased significantly(P<0.05).3.5Effects of Zuogui Pill,Yougui Pill and Their Prescriptions on m RNA and Protein Expressions of C/EBP? in BMSCs of POMP Rats.The results show that,compared with CON group,SHAM and YGW group do not have significantly statistical differences on m RNA expressions of C/EBP?(P>0.05);m RNA expressions of C/EBP?in BMSCs of OVX,ZGW,GTY,ZYY,BYY and BJL group increase significantly(P<0.05).Compared with that in OVX group,in SHAM,ZGW,GTY and BYY group,m RNA expressions of C/EBP? in the BMSCs can be decreased significantly(P<0.05),those in GTY and BYY group can be increased significantly(P<0.05).Compared with that of ZGW group,m RNA expressions of C/EBP?in SHAM group can be decreased significantly(P<0.05),and those in YGW,BYY and BJL group increase(P<0.05).Compared with CON group,SHAM,YGW,GTY and BJL group do not have significantly statistical differences on protein expressions of C/EBP?(P>0.05),in OVX group,proteinexpressions of C/EBP?in the BMSCs increase significantly and the differences have statistical significances(P<0.05).Compared with OVX,YGW,ZYY and BYY group do not have significantly statistical differences on protein expressions of FABP4(P>0.05),in SHAM,ZGW,GTY and BJL group,proteinexpressions of C/EBP?in the BMSCs decrease significantly,and the differences have statistical significances(P<0.05).Compared with that in ZGW group,in YGW,GTY,ZYY,BYY and BJL group,proteinexpressions of C/EBP?in the BMSCs increase significantly,and the differences have statistical significances(P<0.05).3.6Effects of Zuogui Pill,Yougui Pill and Their Prescriptions on m RNA and Protein Expressions of C/EBP? in BMSCs of POMP Rats.The results show that,compared with CON group,SHAM and BJL group do not have significantly statistical differences on m RNA expressions of C/EBP?(P>0.05);m RNA expressions of C/EBP?in BMSCs of OVX,ZGW,YGW,GTY,ZYYand BYYgroup increase significantly(P<0.05).Compared with that in OVX group,in SHAM,ZGW,BYY and BJL group,m RNA expressions of C/EBP?in the BMSCs can be decreased significantly(P<0.05),those in BYY group can be increased significantly(P<0.05).Compared with that in ZGW group,in SHAM,ZYY and BYY group,m RNA expressions of C/EBP?in BMSCs increase(P<0.05),those in YGW,GTY and BJLgroupdo not have significantly statistical differences(P>0.05).Compared with CON group,SHAM and GTY group do not have significantly statistical differences on protein expressions of C/EBP?(P>0.05),in OVX,ZGW,YGW,ZYY and BYY group,protein expressions of C/EBP?increase significantly,and the differences have statistical significances(P<0.05).Compared with that in OVX group,in SHAM,ZGW,YGW,GTY and BJL group,protein expressions of C/EBP ? decrease significantly,and the differences have statistical significances(P<0.05),in BYY group,protein expressions of C/EBP?increase significantly,and the differences have statistical significances(P<0.05).Compared with ZGW group,GTY do not have significantly statistical differences on protein expressions of C/EBP?(P>0.05),protein expressions of C/EBP?in SHAM and BJL group decrease significantly,and the differences have statistical significances(P<0.05),those in YGW,ZYY and BYY group increase significantly,and the differences have statistical significances(P<0.05)Conclusion:1 Zuogui Pill,Yougui Pill and their prescriptions canall enhance the proliferation of BMSCs of the POMP Rats2 After 10 days of adipogenic induction,the expressions of lipid droplets of BMSCs are regulated by Zuogui Pill,Yougui Pill and their prescriptions,indicating that Zuogui Pill,Yougui Pill and their prescriptions have effects on the regulation of adipogenic differentiation of BMSCs.3 Zuogui Pill,Yougui Pill and their prescriptions can regulate theadipogenic differentiation of BMSCs.And the ability of reducing the adipogenic differentiation of ZGW is better than that of its prescription.It proves that the ability of reducing the adipogenic differentiation of Zuogui Pill both nourishing kidney Yin and warming kidney Yang is much better than that of prescription which can only warm kidney Yang or nourish kidney Yin.5 Yougui Pill can improve the ability of theadipogenic differentiation of BMSCs,probably because that Yougui Pill which warms kidney Yang promotes the movement of adipocytes in the bones,and promotes the flow,transformation and formation of fat. |
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