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 observe the changes in morphology, structure, and ventricular function of infarct heart after bone marrow mononuclear cells (BMMNC) implantation. Methods Twenty-four dogs were divided into four groups with random number table, acute myocardial infarction (AM I) control group , AM I-BMMNC group , old myocardial infarct ion (OMI) control group and OM I-BMMNC group , 6 dogs each group. Autologous BMMNC were injected into infarct and peri-infarct myocardium fo r transplantation in AM I-BMMNC group and OM I-BMMNC group. The same volume of no-cells phosphate buffered solution (PBS) was injected into the myocardium in AM Icontrol group and OM I-control group. Before and at six weeks of cell t ransplantation, ult rasonic cardiography (UCG) were performed to observe the change of heart morphology and function, then the heart was harvested for morphological and histological study. Results U CG showed that left ventricular end diastolic dimension (LV EDD) , left ventricular end diastolic volume (LVEDV ) , the thickness of left ventricular postwall (LVPW ) in AM I-BMMNC group were significantly less than those in AM I-control group (32. 5±5. 1mm vs. 36. 6±3. 4mm , 46. 7±12. 1m l vs. 57. 5±10. 1m l, 6. 2±0. 6mm vs. 6. 9±0. 9mm; P lt; 0. 05). LVEDD, LVEDV , LVPW in OM I-BMMNC group were significantly less than those in OM I-control group (32. 8±4. 2 mm vs. 36. 8±4. 4mm , 48. 2±12. 9m l vs. 60.6±16.5m l, 7. 0±0. 4mm vs. 7. 3±0. 5mm; P lt; 0. 05). The value of eject fraction (EF) in OM I-BMMNC group were significantly higher than that in OM I-control group (53. 3% ±10. 3% vs. 44. 7%±10. 1% ). Compared with their control group in morphological measurement, the increase of infarct region thickness (7. 0 ± 1. 9mm vs. 5. 0 ±2.0mm , 6.0±0. 6mm vs. 4. 0±0. 5mm; P lt; 0. 05) and the reduction of infarct region length (25. 5±5. 2mm vs. 32. 1±612mm , 33. 6±5. 5mm vs. 39. 0±3. 2mm , P lt; 0. 05) were observed after transplantation in AM I-BMMNC group and OM I-BMMNC group, no ventricular aneurysm was found in AM I-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OM I-BMMNC group (0. 581±0. 013 vs. 0. 566±0.015; P lt; 0. 05). Both in AM I-BMMNC group and OM I-BMMNC group, fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescencew as irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transp lantation both in AMI and in OM I, and significant lymphocyte infiltration in AM I-BMMNC group. Conclusion BMMNC implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and st ructure, and improve neovascularization in histology, and improve the heart function.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the motor function recovery, the expression of vascular endothel ial growth factor (VEGF) gene, and angiogenesis after spinal cord injury (SCI) in rats, and to explore the treatment mechanism of BMSCs in SCI. Methods BMSCs were isolated and cultured from the marrow of 5 Wistar rats (4 weeks old) and the 3rd-4th passage cells were prepared for the experiment. Atotal of 87 adult female Wistar rats (weighing 220-250 g) were randomly divided into 3 groups: sham-operated group (group A, n=21), DMEM group (group B, n=33), BMSCs group (group C, n=33). A laminectomy was only performed at T8-10 levels in group A. The SCI models were establ ished by modified Nystrom’s compression method in groups B and C, and BMSCs and DMEM were injected in groups B and C respectively at 30 minutes after SCI. Basso-Beattie-Bresnahan (BBB) score was used for the motor function recovery at 3, 7, 14, and 28 days, RT-PCR for the VEGF mRNA at 1, 3, and 5 days, and immunohistochemical staining for angiogenesis at 3, 7, 14, and 28 days. Results In groups B and C, the hindl imb locomotor function was improved at different degrees with time, showing significant difference in BBB score between groups B, C and group A (P lt; 0.05). At 28 days, the BBB score in group C was significantly higher than that in group B (P lt; 0.05) and there was no significant difference between groups B and C (P gt; 0.05) at 3, 7, and 14 days after transplantation. The numbers of microvessels in the ventral horns of gray matter around SCI in groups B and C were significantly lower than that in group C (P lt; 0.05) at 3 days, but there was no significant difference at 7, 14, and 28 days after transplantation (P gt; 0.05). There was no significant difference in the number of microvessels between group C and group B (P gt; 0.05) at 3 and 7 days, but the number of microvessels in group C was significantly higher than that in group B (P lt; 0.05) at 14 and 28 days after transplantation. However, there was no significant difference in the number of microvessels in the white matter around SCI in 3 groups at different time points after transplantation (P gt; 0.05). The RT-PCR results showed that VEGF mRNA expressed at a low level in group A. Compared with group A, the expression level of VEGF mRNA in groups B and C increased at 1 day and reached the peak at 3 days, then decreased at 5 days after transplantation; and the expression of VEGF mRNA was significantly higher in groups B and C than in group A (P lt; 0.05),and in group C than in group B (P lt; 0.05) at 1, 3, and 5 days. Conclusion BMSCs may promote the motor function recoveryby up-regulating VEGF mRNA expression and increasing angiogenesis in the spinal cord after SCI in rats.
Objective To investigate the dose-dependent relationship of bone marrow mesenchymal stem cells(MSCs) transplantation in improving ischemic myocardial dysfunction? in a rat ischemic heart model. Methods Myocardial infarction was induced in 32 inbred F344 rats by acute ligation of the left anterior descending(LAD) coronary artery. One week after ligation, the ratswere randomized? into four equal groups, with eight rats in each group. Equal volume Iscove’s modified Dulbecco’s medium was injected in the control group, 1×103(group 1), 1×105(group 2), and 1×107(group 3) 5-bromodeoxyuridine (BrdU) labeled bone marrow MSCs were injected into the infarcted myocardium. Cardiac function was evaluated by ultrasound before the ligation of the LAD, before the transplantation and the 4th week after transplantation. The expressions of BrdU,Connexin43,Myosin heavy chain β(MHC), and smooth muscle actin α(α-SMA) were detected by immunofluorescence and immunohistochemistry at the 4th week after transplantation. The amount of functional vessels stained by α-SMA was counted simultaneously. Results At the 4th week? after transplantation, the ejection fraction(EF) in goup 2 was more significantly improved than that in group1(0.54±0.20 vs. 0.34±0.16, P＝0.004) and EF in group 3 was more significantly improved than that in group 2(0.71±0.24 vs. 0.54±0.20,P＝0.018), whereas no significant difference between group 1 and control group was detected (0.34±0.16 vs. 0.36±0.15,Pgt;0.05). The BrdU labeled MSCs could be found in host myocardium. The number of cells in group 2 by double staining both for BrdU and for MHC observed in ischemic myocardium were significantly more than that in group 1? (323.20±91.62 n/HP vs. 51.75±27.58 n/HP,P＝0.049) and the same was true between group 3 and group 2(409.75±106.65 n/HP vs. 323.20±91.62 n/HP,Plt;0.001), whereas the result of control group was negative.The majority of transplanted cells were found positive staining both for MHC and for Connexin43 in all groups. There were lots of positive staining of α-SMA whose form were partly irregular in ischemic myocardium indicating that there was neovascularization in group1 and control group. More neovascularization in group2 was found than that in group 1 (28.38±12.79 n/HP vs. 22.75±9.07 n/HP, P＝0015) and more neovascularization in group 3 was found? than that in group 2 (35.63±13.27 n/HP vs. 28.38±12.79 n/HP, P＝0.002) . Conclusion Transplanted into infarcted myocardium, bone marrow MSCs may have significant and dose-dependent potential for cardiomyogenesis with functional recovery from myocardial ischemia.
Objective To study the influence of autologous bone mesenchymal stem cells (BMSCs) on myocardial structure and cardiac function after being implantated into acute infarcted myocardial site. Methods Bone marrow was aspirated from the posterosuperior iliac spine of Guizhou Xiang swine. After being isolated, cultured and co cultured with 5 azacytidine, either autologous BMSCs (total cells 2×10 6, experimental group, n =12), or a comparable volume of culture medium (control group, n =12), was injected into the left anterior descending(LAD) branch of coronary artery just distal to the ligation site of the LAD. The same volume of BMSCs or culture medium was injected into several spots in the infarcted myocardium. Echocardiographic measurements were performed three or six weeks after implantation to assess the myocardial structure and cardiac function. Results Left ventricular function, including eject fraction(EF), fractional shortening and wall thickening, were higher in experimental group when compared with control group. The thickness of the ventricular wall and septum was also found increased while the left ventricular chamber size was smaller in experimental group. Conclusion Implantation of BMSCs into the infarcted myocardium is believed to attenuate the remodeling process, inhibit the extent of wall thinning and dilatation of the ventricular chamber. BMSCs implantation may also improve the contractile ability of the myocardium and cardiac function.
Objective To explore the middle-term outcome of autologous bone marrow mononuclear cells transplantation in the treatment of lower l imb ischemia. Methods From March 2003 to June 2005, 65 patients with lower l imb ischemia were treated by autologous bone marrow mononuclear cells transplantation. Of the patients, there were 50 males and 15 females, with a mean age of 66.5 years (range 36-89 years), including 4 cases of simple arteriosclerotic occlusion,5 cases of thromboangiitis obl iterans and 56 cases diabetic lower l imb ischemia. A total of 400 mL bone-marrow blood were extracted from the posterior superior il iac crest. And then the mononuclear cells were isolated from the bone-marrow blood in the laboratory. The amount of transplantation bone marrow mononuclear cells was (0.60-1.80) × 109 (mean 1.05 × 109). Twelve patients received cell transplantation from two to four times and the other patients one time. According to the improvement of cl inical finding, the outcome was evaluated. Results All the patients were followed up for 8-56 months (mean 21.5 months). There were 8 deaths, and the mortal ity was 12.3%; 5 were due to myocardial infarction and heart failure and 3 were due to cerebral infarction. The general effective rate was 70.8% (46/65) and the recurrent rate was 10.7% (7/65). Of them, the response to treatment lasted over 12 months in 42 cases, accounting for 91.3% (42/46); over 24 months in 24 cases, accounting for 52.2% (24/46); and over 37 months in 12 cases, accounting for 26.1% (12/46). The effective rates were 100% in 12 patients who received 2-4 times transplantation and 64.2% in 53 patients who received 1 time transplantation, showing statistically significant difference between them (P lt; 0.001). Conclusion The middle-term outcome of autologous bone marrow mononuclear cells transplantation show that it is a feasible and simple method for treatment of lower l imb ischemia.
Objective To investigate the cl inical effect of MSCs transplantation derived from human umbil ical cord on bone nonunion. Methods From December 2005 to December 2007, 72 patients with traumatic bone nonunion were treated. Auto-il iac bone transplantation was used in 36 patients (group A), including 27 males and 9 females, aging (34.0 ± 2.1) years; including 18 cases of femoral fracture and 18 cases of tibia fracture; and the time of bone nonunion being (9.1 ± 1.7)months. Percutaneous MSCs transplantation derived from human umbil ical cord was used in 36 patients (group B), including 28 males and 8 females, aging (36.0 ± 1.6) years; including 18 cases of femoral fracture and 18 cases of tibia fracture; and the time of bone nonunion being (6.4 ± 1.9) months. There were no statistically significant differences in general data between two groups (P gt; 0.05). In group A, the site of bone nonunion was filled with relevant auto-il iac bone. In group B, the mixture of 6-8 mL platelet-rich plasma prepared by centrifugal izing venous blood and 1 × (106-107) P5 MSCs extracted from human umbil ical cord denoted by volunteers was injected into the region of bone nonunion with 0.2 g demineral ized bone powder. Results Incision healed by first intention in group A. No puncture, deep infection, rejection and general fever reaction occurred in group B. All patients in two groups were followed up for (13.2 ± 4.6) months. No loosening and breakage of internal fixation were observed in two groups. The motil ity and function of hip, knee and ankle were good. The time of bone union was (10.3 ± 2.8) months in group A and (5.6 ± 0.8) months in groups B, showing significant difference between two groups (P lt; 0.05). Conclusion The percutaneous MSCs transplantation derived from human umbil ical cord is more effective on bone nonunion than the traditional treatment, it is easily-to-operate, safe, rel iable, and rapid for union. It is one of effective methods in treating bone nonunion.
Objective The human amniotic epithel ial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for l iver regeneration. To research the survival and migrationin vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and toexplore the expression of hepatocyte-l ike function. Methods Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal sal ine was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of l iver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. Results No tumor tissue was found in l iver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundl ike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the l iver of group A; no abnormal cells were found in l iver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and l iver of group A with expressing human albumin, but no GFP+ cells was found in l iver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-l ike function. It will be beneficial to further identify their biological characteristics.
Objective To investigate effect of intravitreal injection of FK506 on the survival of human retinal pigment epithelial (RPE) cells heter oplastically transplanted into the subretinal space of rabbits.Methods The immortalized human RPE cells were genetically labeled by retrovirus vector carrying a green fluorescent protein (GFP). A total of 50 μl RPE cells suspension with 4×103 cells/μl which expressed GFP were injected into the subretinal space of both eyes of 18 white rabbits and 10 gray rabbits. The left eyes of all of the rabbits were injected of 5 μl FK506 (5 μg/μl) intravitreally once a week during the first 5 weeks, then once every other week until the 20th week and the right eyes were as the control. The histological sections of heteroplastic RPE cells were observed by epifluorescent microscope.Results GFP-expressing cells could be seen after 1 week, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 26, 33, and 54 weeks in white rabbits and after 4 , 5, 6, 7, 14, 18, 20, and 26 weeks in gray rabbits. The configuration and integrality of the RPE-GFP cells in the left eyes which had been intravitreally injected of FK506 1-14 weeks after transplantation were better than those in the right eyes without injection. After 18 weeks, the condition of heteroplastic cells with few difference in both eyes in 7 white and 3 gray rabbits were found. After 1-6 weeks, focal and disseminated lymphocytes around the choroidal small vessles of right eyes in 6 white and 3 gray rabbits could be seen while the infiltration of the lymphocytes in the left eyes was much reduced.Conclusion Intravitreal injection of a small amount of FK506 at the first 3 months after transplantation may significantly improve the survival of heteroplastic RPE cells in the subretinal space of rabbits. (Chin J Ocul Fundus Dis，2003，19：333-404)
Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells (BMSCs) in injured spinal cord by subarachnoid administration, and to evaluate the most optimal subarachnoid administration times for BMSCs. Methods Eight adult male rats (weighing 120 g) were used to isolate BMSCs that were cultured, purified and labeled with Hoechst 33342 in vitro. Another 75 adult Wistar rats (weighing 220 g) were made the spinal cord injury (SCI) models at T9,10 level according to the improved Allen’s method and were randomly divided into 5 groups (groups A, B, C, D, and E, n=15). The labeled BMSCs at 1 × 107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A (one time at 1 week), B ( two times at 1 and 3 weeks), C (3 times at 1, 3, and 5 weeks) and D (5 times at 1, 3, 5, 7, and 9 weeks) and 0.2 mL phosphate-buffered sal ine (PBS) was injected in group E (5 times at 1, 3, 5, 7, and 9 weeks) as blank control. The neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) scale 1, 3, 5, 7, 9, and 12 weeks after transplantation. The migration, survival, differentiation, and histomorphological changes of BMSCs were observed by HE, immunohistochemistry, and fluorescence microscopy. Results At 3 weeks after injury, there were significant differences in the BBB scores between group E and groups A, B, C, D (P lt; 0.01), and between groups A, B and groups C, D (P lt; 0.01). At 7, 9, and 12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P lt; 0.01), and in group B than in group A (P lt; 0.01). There were no significant differences in the BBB scores between groups C and D (P gt; 0.05). The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the transplanted cells gradually decreased in group A; in groups B, C, and D, BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B (P lt; 0.01). HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C, and D. At 12 weeks, the area of cavity was the miximal in groups C and D, moderate in groups A and B, and the maximal in group E. The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B. The expression of NF-200-positive fibers was more intense in group C. Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions, and three times for BMSCs transplantation is best