| BackgroundWith the improvement of people’s living standards and changes in diet, theincidence of coronary artery disease (CHD) had been ascending year by year.The short and long-term complications of myocardial infarction not onlythreatened human life, but also seriously affected people’s living quality.Although many researches had shown that some cardiomyocyte cells aftermyocardial infarction could split and proliferate, only1%of cells updating ratewas difficult to compensate for the loss of the cardiomyocyte cells or had littleamelioration on heart function. The clinical treatment of myocardial infarctionfocused on the rescue of non-infarcted cardiomyocyte cells and theimprovement of heart function, but drug therapy, percutaneous transluminalcoronary angioplasty or coronary artery bypass grafting couldn’t be able torestore the infarcted heart function completely. So the mortality in patients who suffered with severe myocardial infarction remained rather high. Repairment orreplacement of the infarcted cardiomyocyte cells became the great challengeswhich clinical doctors and scientists have to solve. In recent years, manyresearches showed that the cardiomyocytes differentiated from stem cells couldreplace infarcted cardiomyocytes cells and improve heart function, which havegiven us hopes to further treat coronary heart disease. Stud ies of bone marrowmesenchymal stem cells (BMMSCs) transplantation in treatment of myocardialinfarction have made chanllenging progresses, but how to get sufficient numberof cardiomyocyte-like cells derived from BMMSCs have been kept as one hotand difficult spots. As research progressed, difficulties in research of ischemicheart disease would be resolved gradually in the near future and BMMSCstransplantation for treatment of myocardial infarction would obtain significanttherapeutic effects for patients.Objectives1. Study on rat BMMSCs isolation, cultivation and purification methods invitro.2. Exploration of the experimental methods of differentiation of BMMSCsinto cardiomyocyte-like cells induced by BMP-2.3. Exploration of the effections of differentiation of BMMSCs intocardiomyocyte-like cells induced by BMP-2and5-AZA.Methods1. The rat BMMSCs isolation and culture in vitro: After preparation of ratbone marrow of long bones of limbs, using density gradient centrifugationcombined with differential attachment method to separate the BMMSCs, thensubculture cells for purification. The fourth generation of BMMSCs was detected by flow cytometry for surface antigens CD29, CD44and CD45.Observation the dynamic growth with inverted phase contrast microscope andrecord pictures.2. BMMSCs differentiation into cardiomyocyte-like cells induced by BMP-2:Fourth generation of BMMSCs induced by BMP-2and changed medium24hlater. Experimental group seted as blank control group (Control) and BMP-2group (10μg/L). Western Blot detected connexin-43(CX43) and cardiac troponinI (cTn I) expression of2w and4w. Immunofluorescent staining is used foridentification of cTn I and CX43expression of cardiomyocyte-like cells inducedfor4w by BMP-2.3. BMMSCs differentiation into cardiomyocyte-like cells induced by5-AZAand BMP-2: The fourth generation of BMMSCs were divided experimentalgroups as follows: blank control group (Control),5-AZA group (10μmol/L),BMP-2group (10μg/L), BMP-2+5-AZA group (10μg/L+10μmol/L). Theformer3groups were changed with fully cultured medium after induced for24hand the combined group was changed with fully cultured medium after inducedfor total48h. Living cells percentages when induction termination weredetected by flow cytometry.The expressions of CX43and cTn I of each groupswere identificated by immunofluorescent staining cells after cultured for4w;the expressions of cTn I and CX43were detected by Western Blot;differentiation rate of BMMSCs were detected by flow cytometry.Results1.Morphological characteristics of BMMSCs cultured in vitro: primarypasspage of BMMSCs had small cell nuclear and variety of forms, includingmainly short shuttle-shaped, oval, star-shaped or irregularly-shaped, and withthe varying length of protruding out of the cell body. After1w, cell growth was colony-like and could be found the "whirlpool-like" structure. Cells bulked upafter the passage and arranged in irregular uneven shape, with largerkaryoplasmic ratio and arrangement disorder. Flow cytometry identificationresults for BMMSCs surface antigens: CD29positive expression rate was (94.4±3.59)%; positive expression rate of CD44is (88.4±1.51)%; expression ofCD45positive rate is (2.6±0.40)%.2. BMMSCs differentiation into cardiomyocytes induced by BMP-2:Oberserved morphological changes in the cells under the inverted phase contrastmicroscope. Control group without obvious changes; cell volume and thekaryoplasmic ratio of BMP-2group increased, with the shape of cells were longfusiform and growth direction arranged consistent with the same over time.Cell-cell junction became close and appeared "muscle island-like" structure in4w. Immunofluorescence staining show that cTn I and CX43expression could befound in BMP-2cells after induced for4w, but none of cell expressed cTn Iand only a little of cells expressed CX43in Control group. Western Blot wasused for detection of protein expression levels: BMP-2group of2w have littlecTn I and CX43expression, and which of4w is significantly higher than2w.3. BMMSCs are induced to differentiate into cardiomyoctye-like cells byBMP-2and5-AZA: When induction was terminated, the percentages of livecells were detected by flow cytometry. Results have shown that (45.23±5.73)%of total cells living in5-AZA group,(73.56±3.78)%of total cellsliving in BMP-2group,(68.75±6.82)%of total cells living in BMP-2+5-AZAgroup. Morphological changes of cells under the inverted phase contrastmicroscope showed that: no obvious changes were observed in control groupcells; cell morphology of other groups was significantly changed: nucleusbecame large and cells formed long spindle-shaped. Connections between cells grew tightly, and cells growth tended to concentrate and form "muscleisland-like" structure in4w. The result of immunofluorescence staining showedthat after induction of2w, none of cTn I expression could be seen in controlgroup and a little expression could be seen in other three groups. In controlgroup there was still no expression after induction for4w, but cTn I expressionof other three groups were significantly enhanced. Western Blot showed thatafter induced for2w, control group had weak expression and other three groupsexpressed weak. After induced for4w, control group still had weak expression,but cTn I and CX43expression of the other three groups greatly enhanced.Compared with control group, each of other three groups had significantlystatistics differences (P<0.01); comparisons between combined induction group,5-AZA group and BMP-2group had statistics differences (P<0.05); proteinexpression of2w and4w had statistical differences in three groups (P<0.05).Differentiation rates of cardiomyocyte-like cells were detected by flowcytometry and the results displayed that in control group FITC marked ofpositive cell counted number for (2.05±0.39)%;5-AZA group FITC marked ofpositive cell counted number for (23.78±1.53)%; BMP-2group FITC markedof positive cell counted number for (17.53±1.06)%; BMP-2+5-AZA groupFITC marked of positive cell counted number for (27.99±2.01)%.5-AZAgroup, BMP-2group and the BMP-2+5-AZA group had significant statisticaldifferences compared with the control group (P<0.01).Conclusion1. The differentiation rate of BMMSCs induced by BMP-2and5-AZA was(27.99±2.01)%, and cell death significantly lower than that of induced by5-AZA alone. BMP-2combined with5-AZA could be a safe and effectivemethod of differentiation. 2.The differentiation rate of BMMSCs induced by BMP-2is (17.53±1.06)%,BMP-2could induce BMMSCs differentiated into cardiomyocyte-like cellswhich show the specific biological characteristics, but the differentiation ratewas lower than induced by5-AZA alone.3. BMMSCs could be isolated by using density gradient centrifugationcombined with differential attachment method, the surface antigen of4thgeneration BMMSCs was detected by flow cytometry, which confirmed thathigh purity BMMSCs have been obtained. |
PDF Full Text Request