Effect of bone marrow stromal cells combined with pLXSN-bcl-2 gene on rat cerebral ischemia

Aim: To discuss the therapeutic effect of grafting bone marrow stromal cell (MSC) and plasmid pLXSN2-mediated transfer of bcl-2 gene to treat local cerebral ischemia in rats. Methods: The experiment had been completed at the China Medical University from May 2005 to October 2006. 1 Forty Wistar female rats were randomly divided into control group, cell transplantation group, gene plasmid group and combination group, each contained 10 rats. Ten wistar male rats were adopted to extract the MSC. Plasmid pLXSN-bcl-2 gene was offered by Doctor Qian Qi-jun. 2 bone marrows were isolated from rat humerus, tibia and femur sterilely, followed by removing metaphysis. And the cell suspension after centrifugation was used for primary culture. When reaching 80% confluence, the cells covering the bottom of culture bottle were digested using trypsin and passaged to the third generation at the density of 1×10 4/CM 2 for transplantation, 24 hours before which the cells were labeled with BrdU. Then plasmid pLXSN-bcl-2 was amplificated, extracted, identified and purified for preservation at -20 °C. The ischemia-reperfusion rat models were established by the transient occlusion of left middle cerebral artery using modified thread, 3 hours later the plasmid was injected into rat carotid artery at the total dose of 10 μg and total volume of 200 μL. Moreover 1 mL suspension containing 3×10 6 MSC was injected into the tail vein of rats. While equal volume of physiological saline was given in the model control group. 3 Neurological Deficits Score (NDS) was observed in all the conscious rats at days 3 and 14 after transplantation. Then the cerebral tissues were obtained after the rats were anesthetized to death. Double-staining immunohistochemistry of brain sections was used to identify brain-derived neurotrophic factor (BDNF), Bcl-2 protein expression, the distribution and quantity in the brain of the BrdU-labeled MSC. TUNEL method was employed to detect the cell apoptosis. Results: 1 NDS in the combination group of 3 days postoperation was significantly lower than that in the model control group, and also significantly lower than other groups (P < 0.05). 2 BrdU-positive MSC was observed around the ischemic brain tissues, then hippocampus and corpora striatum, additionally the contralateral area of infarction appeared a few coils. BrdU, BDNF and bcl-2 protein expressions in brain tissues of the combination group were higher than those in other 3 groups (P < 0.05). Conclusion: MSC and pLXSN-bcl-2 gene combined transplantation has the obvious therapeutic effects in rats with cerebral ischemia, and the effect is superior to the simple administration. Its mechanism possibly is that bcl-2 gene can reduce both the apoptosis of grafted MSC and the cellular apoptosis in the brain.