ObjectiveTo evaluate the effect of leucocyte- and platelet-rich plasma (L-PRP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in treating avascular necrosis of the femoral head (ANFH) in rabbits. MethodsTwenty-four New Zealand white rabbits (4-6 months old, both genders, weighing 2.0-3.0 kg) were used for the establishment of bilateral ANFH models and divided into 4 groups (n=6). BMSCs were isolated from the bone marrow of iliac crest, cultured and identified. L-PRP was prepared by Landesberg method. Core decompression only (group A), core decompression and L-PRP implantation (group B), core decompression and BMSCs implantation (group C), and core decompression and implantation of BMSCs and L-PRP were performed in 4 groups. To evaluate bone formation and remodeling of the defects, X-ray photography was taken at 2, 4, and 8 weeks postoperatively. The modified Lane-Sandhu scoring system was used to evaluate the bone formation. Two rabbits were sacrificed at 2, 4, 8 weeks after operation to harvest the specimens for histological observation, new blood vessel count and new bone area ratio. ResultsThe observations of radiology and histology displayed different degrees of bone regeneration at bone defect sites in each group. At 2, 4, and 8 weeks postoperatively, the results of Lane-Sandhu X-ray photography scoring, new blood vessel count, and new bone area ratio showed that groups C and D were significantly better than groups A and B, group D was significantly better than group C. and group B was significantly better than group A (P<0.05). ConclusionThese findings demonstrate that L-PRP can promote osteogenic differentiation of BMSCs in treating ANFH in rabbits, and core decompression associated with BMSCs and L-PRP is an effective and feasible method to treat ANFH.
ObjectiveTo explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. MethodsBMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1×10-10 mol/L group, OGP 1×10-9mol/L group, OGP 1×10-8 mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10%FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. ResultsBMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1×10-8 mol/L and 1×10-9 mol/L groups was significantly higher than that of the control group in all serum concentrations (P<0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1×10-8 mol/L group was significantly higher than that of the other 2 OGP groups (P<0.05), but when FBS concentration was 10%, OGP 1×10-8 mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P<0.05). Under the condition of 5%FBS and 8%FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1×10-8 mol/L group (P<0.05). Under the condition of 10%FBS, the ALP activity of each OGP group was still greater than that of the control group (P<0.05), but no significant difference was found between the OGP 1×10-8 mol/L group and OGP 1×10-9 mol/L group (P>0.05). ConclusionThe concentration of 8%FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1×10-8 mol/L.
Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
【Abstract】 Objective To investigate the effect of allogeneic bone marrow-derived mesenchymal stem cells ( BMSCs) transplantation on the airway inflammation and airway remodeling in chronic asthmatic mice. Methods Forty female BALB/c mice were equally randomized into four groups, ie. a normal control group, a BMSCs control group, an asthma model group, and a BMSCs transplantation group. BMSCs were generated from male donor mice, then the mice in the asthma model group and the BMSCs transplantation group were sensitized and challenged with OVA to establish chronic asthmatic mice model. Hematoxylin and eosin staining and Alcian blue-periodic acid-Schiff staining were used to analyze the effects on airway inflammation and airway remodeling after BMSC engraftment. The number of CD4 + CD25 + regulatory T cells in spleen was detected by flow cytometry. Results In lungs of the asthmamodel group, there were intensive inflammatory cells infiltration around airway and blood vessels, goblet cell proliferation, epithelial desquamation, patchy airway occlusion by hyperviscous mucus, and hypertrophy of airway smooth muscle.Airway inflammation and airway remodeling were significantly relieved in the BMSCs transplantation group.There was no obvious inflammatory cells infiltration in the airway and airway remodeling both in the normal control group and the BMSCs control group. The number of CD4 + CD25 + regulatory T cells in spleensignificantly decreased in the asthma model group compared with the two control groups ( P lt; 0. 05) , and significantly increased in the BMSCs transplantation group compared with the asthma model group ( P lt;0. 05) . There was no significant difference in the number of CD4 + CD25 + regulatory T cells in spleen betweenthe control groups and the BMSCs transplantation group. Conclusion BMSCs engraftment can up-regulate CD4 + CD25 + regulatory T cells and relieve airway inflammation and airway remodeling in asthmatic mice.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To explore the induction of cardiomyogenesis of microRNA-129 （mir-129） in rat bone marrowmesenchymal stem cells （BM-MSCs） and its mechanism. Methods BM-MSCs were isolated from Sprague-Dawley rats and cultured in vitro. Overexpression of mir-129 or both mir-129 and glycogen synthase kinase-3β （GSK-3β） in BM-MSCs was produced with a lentiviral vector system. All the BM-MSCs were divided into four groups: control group （MSCs），Lentiviral vectors＋MSCs group （Lv-MSCs），mir-129 transfection group （mir-129-MSCs），and mir-129＋GSK-3βdouble transfection group （mir-129＋GSK-3β-MSCs）. Five-Azacytidine （5-Aza） （10 μmol/L） was used to induce BM-MSCsdifferentiation into cardiomyocytes. On the 1st，5 th，10 th，15 th and 20 th day after induction，realtime-PCR was performedto detect mRNA levels of GATA-4，Nkx2.5 and MEF-2C. On the 10 th，15 th and 20 th day after induction，Western blottingwas performed to examine expression levels of cTnI，Desmin，GSK-3β，phosphorylated β-catenin and dephosphorylated β-catenin. Results Compared with the control group，at respective time points，mRNA levels of cardiomyogenic genes and expression levels of cardiomyocyte-related proteins of mir-129 transfection group were significantly elevated，theexpression level of GSK-3β was significantly decreased，and the ratio of dephosphorylated/phosphorylated β-catenin was significantly elevated. When both mir-129 and GSK-3β were overexpressed in BM-MSCs，mRNA levels of cardiomyogenicgenes and expression levels of cardiomyocyte-related proteins were significantly lower than those of mir-129 transfection group，and the ratio of dephosphorylated/phosphorylated β-catenin was significantly decreased. Conclusion Overexpression of mir-129 can promote cardiomyogenesis of rat BM-MSCs possibly via inhibiting GSK-3β production and thus decreasing the inhibition of phosphorylation of β-catenin which then enters the nucleus and activates downstream signaling pathways that regulate cardiomyogenic differentiation of BM-MSCs.
Abstract： Objective To study the effect of different numbers of bone marrow mesenchymal stem cells（MSCs） transplanted into rats with pulmonary arterial hypertension （PAH）induced by monocrotaline（MCT）and their influence on the expression of endothelin-1（ET-1）. Methods Forty healthy male Wistar rats（weight，from 180 to 250 g） were divided into four groups by random number table（n=10）：group A：Wistar rats were intraperitoneally injected with MCT 60 mg/ kg， and then injected with 1×106 MSCs via the external jugular vein；group B：Wistar rats were intraperitoneally injected with MCT 60 mg/kg，and then injected with 5×105 MSCs via the external jugular vein；MCT group：Wistar rats were intraperitoneally injected with MCT 60 mg/kg， and then injected with equal amount of PBS via the external jugular vein； control group：Wistar rats were intraperitoneally injected with equal amount of saline and then injected with equal amount of PBS via the external jugular vein. Four weeks after MSCs transplantation，right ventricular systolic pressure（RVSP） and ventricular weight ratio of right ventricle/ （left ventricle＋ventricular septum）were measured. Histomorphology of lung tissue was observed. Genetic expression of ET-1 in lungs and serum peptide of ET-1 were also measured. Results Four weeks after MSCs transplantation，both RVSP and ventricular weight ratio decreased significantly in rats of group Acompared with those of MCT group（RVSP：35.8±4.2 mm Hg vs. 47.2±10.1 mm Hg，P＜ 0.01； ventricular weight ratio：0.357±0.032 vs. 0.452±0.056，P＜0.01）， but these two parameters didn’t decrease significantly in rats of group B（P＞ 0.05）. By histopathological staining， the percentage of medial wall thickness of the pulmonary arterioles was significantly less in rats of group A than that of MCT group（19.7%±3.0% vs. 26.8%±3.6%， P＜ 0.01）. There was no statistical difference in the percentage of medial wall thickness of the pulmonary arterioles between group B and MCT group. Reverse transcriptase-polymerase chain reaction （RTase-PCR）results showed that ET-1messenger ribonucleic acid（mRNA）expression was highest in MCT group and MSCs transplantation significantly decreasedits expression in group A， while its expression was similar between group B and MCT group. The expression ofET-1 in plasma was also significantly decreased in group A than that in MCT group. Conclusion Intravenous MSCs transplantation can significantly inhibit MCT-induced PAH，and reduce both ET-1 mRNA expression in lung and ET-1 peptide level in plasma. It’s a better choice to transplant 1×106 MSCs to inhibit PAH in rats.
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
Abstract： Objective To construct a nesprin-siRNA lentiviral vector（LV-siNesprin）, transfect it into bone marrow mesenchymal stem cells （MSCs）, and observe morphology changes of MSCs. Methods According to the target gene sequence of nesprin, we designed and synthesized four pairs of miRNA oligo, which were then annealed into double-strand DNA and identified by sequencing. MiRNA interference with the four kinds of plasmids （SR-1,SR-2,SR-3, andSR-4） were transfected into rat vascular smooth muscle cells, and reverse transcriptase chain reaction(RT-PCR) and Western blotting were performed to detect the interference effects and filter out the most effective interference sequence. We used the best interference sequence carriers and pDONR221 to react together to get the entry vectors with interference sequence. Then the objective carrier pLenti6/V5-DEST expressing both entry vectors and lentiviral vectors was restructured to get lentiviral expression vector containing interference sequence （LV-siNesprin＋green fluoresent protein （GFP））, which was packaged and the virus titer was determined. LV-siNesprin＋GFP was transfected to MSCs, and the expression of nesprin protein（LV-siNesprin＋GFP group，GFP control group and normal cell group）was detected by Western blotting. The morphology of MSCs nuclear was observed by 4’,6-diamidino-2-phenylindole （DAPI） stain. The proliferation of MSCs （LV-siNesprin＋GFP group，GFP control group and normal group） was detected by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide（MTT） after lentivirus transfected to MSCs at 24, 48, 72, and 96 hours. Results The four pairs of miRNA oligo were confirmed by sequencing. Successful construction of LV-siNesprin was confirmed by sequencing. The best interference with miRNA plasmid selected by RT-PCR and Western blotting was SR-3. Lentiviral was packaged, and the activity of the virus titer of the concentrated suspension was 1×106 ifu/ml. After MSCs were transfected with LV-siNesprin, nesprin protein expression significantly decreased, and the nuclear morphology also changed including fusion and fragmentation. The proliferation rate of MSCs in the LV-siNesprin＋GFP group was significantly slower than that of the GFP control and normal cell groups by MTT. Conclusion Nesprin protein plays an important role in stabilizing MSCs nuclear membrane, maintaining spatial structure of MSCs nuclear membrane，and facilitating MSCs proliferation.
Objective To investigate the effect of bone marrow mesenchymal stem cell (MSCs) transp1antation combined with transmyocardial drilling revascularization (TMDR) and degradable stent on myocardium revascu1arization after acute myocardial infarction(AMI), and to provide the experimental evidence for surgical treatment of myocardial infarction. Methods After established models of AMI, the 24 pigs were divided into four groups with random number table, 6 pigs each group. Control group: only established models of AMI; MSCs group: AMI immediately followed by MSCs implantation; TMDR combined with stent group: AMI followed by TMDR and absorbable basic fibroblast growth factor (bFGF) stent implantation; MSCs combined with TMDR and stent group: AMI followed by TMDR and absorbable bFGF stent implantation, and then MSCs implantation. Three months after operation, the infarcted areas and vessel density in infarcted zone were detected by histopathology method. Results Three months after operation, the histopathological examination showed that infarcted areas in MSCs group, TMDR combined with stent group, and MSCs combined with TMDR and stent group were decreased as compared with control group （27.9%±3.1% vs. 48.9%±2.7%，P=0.000；20.3%±1.7% vs. 48.9%±2.7%，P=0.000；12.5%±1.9% vs. 48.9%±2.7%，P=0.000）; and vessel density was further increased （8.4±1.2/HP vs.4.5±14/HP，P=CM(1583mm] 0.001；11.5±2.6/HP vs.4.5±1.4/HP，P=0.001;15.6±1.4/HP vs.4.5±1.4/HP，P=0.000）. Conclusion [CM)]MSCs transplantation combined with TMDR and absorbable bFGF stents implantation could significantly reduce the infarction areas, increase the vessel density. This method may enhance the efficacy of MSCs transplantation in acute cardiac infarction model, which provide a new ideas for the surgical treatment of myocardial infarction.