Objective To observe the changes in the number and function of bone marrow-derived endothel ial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemicl imb disease after bone-marrow stimulation through autologue bone-marrow stem cell implantation. Methods Twelvemale Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n=6) and the control group(n=6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocytemacrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labbled acetylated low density l ipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive abil ity of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindl imb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n=6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA). Results After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 ± 37.0)/HP and (95.2 ± 39.4)/HP, respectively, and there was significant difference between the two groups (P lt; 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 ± 4.3)/HP and (15.0 ± 5.2)/HP, respectively, and the difference between the two groups was significant (P lt; 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 ± 1.2) compared with the control group (2.7 ± 0.8), (P lt; 0.05). Conclusion Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologue bone-marrow stem cell implantation improves the curative effect of ischemic l imb diseases after bone-marrow stimulation.
Objective To investigate the effects of tissue inhibitor-3 of matrix metalloproteinases(TIMP-3) genetransfected vascular smooth muscle cells(VSMCs) transplantation on heart structure after acute myocardial infarction (AMI) in rats and to explore the potential mechanisms. Methods Sixty-one female Wistar rats were produced AMI models by ligating the descending left coronary artery. Fifty-four rats were survived and divided into 3 groups randomly(n=18): 0.5 ml PBS containing 1×106 TIMP-3 gene-transfected VSMCs(group A), 1×106 VSMCs(group B) or 0.5 ml PBS without cell(group C) were injected into the ischemic myocardium immediately. Ischemic myocardium samples were harvested at 1 weekafter operation. The heart structure was observed through the tissue morphologic examination. The activity of TIMP-3 gene-transfected VSMCs were measured by immunohistochemical method. Proteins of TIMP-3 and matrix metalloproteinase 9(MMP-9) were determined by Western blot. Results VSMCs were cultivated and had a high purity(98%). TIMP-3 gene was transfected into VSMCs successfully. One week after operation in groups A, B and C, the average percentage of infarction myocardium size 〖KG6〗and left ventricle free wal area were 28.73%±1.56%, 39.63%±1.84% and 46.32%±2.16% separately.Group A was significantly lower than groups B and C(P＜0.01), group B was significantly lower than group C(P＜0.01). In groups A, B and C the averageleft ventricle volume indexes were 5.27±0.21 mm3／g, 6.69±0.34 mm3／g and 9.67±0.88 mm3／g respectively. Group A was significantly smaller than groups B and C(P＜0.01), group B was significantly smaller than group C(P＜0.01). The immunohistochemical observation confirmed that the implanted VSMCs and TIMP-3 gene were survival in ischemic area. The protein content of TIMP-3 in ischemicmyocardium was significantly higher in group A (300 704.8±3 692.8) than in groups B and C(195 548.8±3 014.2，177 991.1±2 502.1)(P＜0.01), the protein content of MMP-9 in ischemic myocardium was significantly lower in group A(594 827.4±5 708.5) than in groups B and C(921 461.4±8 887.4，1 044 445.0±8 788.6)(P＜0.01). Conclusion Implanted TIMP3 gene transfected VSMCs in ischemic myocardium can conspicuously reduce the myocardium remodeling after AMI.
Purpose To investigate the development of embryonic stem cells (ESC)in the subretinal space． Methods ESC were cultivated in suspension for 4 days till they developed into cell aggregates,i.e.embryonic body(EB).ESC as well as EB combined with or without RA were respectively transplanted into vitreous cavity and subretina1 space in SD rats,and the subretinal transplanted eyes,transient ischemia-reperfusion injuries were made by ligating the ophthalmic artery for 40 seconds before the transplantation .The experimental eyes were enucleated for histological and immunohistochemical assays after 14～28 d. Results The EB was found to develope into photoreceptors induced by RA in the subretinal space under an ischemia-reperfusion condition，and EB transplantation without RA induction induced multiple differentiations in the subretinal space.The single injection of RA without EB induced hyperplasia of the neural retinal cells.ESC transplanted into vitreous cavity rapidly proliferated and developed into atypical hyperplastic mass. Conclusion EB derived from ESC can differentiate into photoreceptors induced by RA in the host subretinal space under an ischemia-reperfusion condition. (Chin J Ocul Fundus Dis，2000，16：213-284)