Study of Intracerebral Transplantation of Umbilical Cord Blood Mesenchymal Stem Cells in a 6-Hydropamine Rat Model of Pakinson’s Disease
|School||Taishan Medical College|
|Keywords||Mesenchymal stem cells Parkinson’s disease transplantation|
Objective To study the theoretical foundation and the feasibility of Umbilical cord blood mesenchymal stem cells （MSCs） transplanted into the Parkinson’s Disease （PD） rats. Methods 1、Establish PD rat model: After anaesthesia by 1% of pentobarbital sodium, Rats were fixed on stereotaxic apparatus and expose the anterior fontanel which was set as zero point. Two points were marked: posterior fontanel 4.4mm, right midline 1.2mm, inferior dura mater 7.8mm; posterior fontanel 4.8mm, posterior fontanel 1.0mm, inferior dura mater 7.8mm. Injected 5μL 6-OHDA containing 0.2 g/L of acrobat into each point by micro-injector. The needle was rotated while inserted with speed of 1μL/minute; Needle was kept in the point for 5 minutes after injection and removed slowly. Sutured skin, injected 20000 U penicillin everyday in case of preventing infection. After surgery at 2 weeks, injected apomorphine （0.05mg/Kg） into the rats. Count the rounds per minute after 30 minutes. The successful PD rat model is the one who rotated over 7 rounds in per minute. At last, we choose Twenty-two PD models from 60 SD rats according to this criterion.2、Separation of MSCs in cord blood: Cord blood samples were obtained from the department of obstetrics and gynecology of the liaocheng people’s hospital which had 60 ml to 120 ml cord blood.3、MSCS separation: The culture medium was abandoned until the above cultured cell had 80% confluence. The cells were then rinsed twice by PBS before digested mixture with 2.5g/L tyrosine and 0.5g/L FCS under 37℃and the digestive process was controlled under microscope. The third generation was selected for transplantation. 4、MSCS transplantation: MSCS which was adjusted to 4.5×106L-1 was transplantation into the PD rat’s models. Rats were selected and allocated randomly into 2 groups:①MSCs group （n=12）:5ul of MSCs was injected.②PBS group: 5ul of PBS was injected. After anesthesia by 1% of pentobarbital sodium, Rats were fixed on stereotaxic apparatus and confirmed the transplantation site in striatum: posterior fontanel 1.0mm, right midline 3.0mm, inferior Dura mater 5.0mm;posterior fontanel 1.0mm, right midline 3.4mm, inferior Dura mater 4.5mm. The injecting method was as above.5、Behavioral identification: After transplantation the rotation tests were conducted every week with the same method that apomorphine was peritoneal injected to induce rotation.6、Immunohistochemical : At 2 weeks and 8 weeks after transplantation, two rats were chosen to expose the heart and conduct through ascending aorta by intubatton by 100ml of physiological saline followed by 150ml of 10% paraform. The brain was taken out and then fixed in the 10% paraform for 24 hours. The samples which embedded with paraffin for serial coronal sectioning in a thickness of 5 um were stained by immunohistochemical staining. Results were observed under optical microscope.7、Statistical analysis: The data from experimental were analyzed with Medical statistical analysis software SPSS 11.0 by the first author, and all the data were expressed with Means, t-test and analysis of variance were used to compare the changes of behaviors between the two groups.RESULTS1. Detection of apomorphine induced rotational behavior in PD rats: Apomoorphine was used to induce rotational behavior every week after transplantation. The rotational rounds of rats significantly reduced from 3 weeks between MSCs groups and PBS groups （P<0.05）.2. Immunohistochemistry: TH-positive cells in the right striatum less than that of left side in MSCs groups. But there was no significantly difference （P<0.05）. No TH-positive cells in the right striatum in PBS groups.3. TH-positive cells in the right SNp in rats of the two groups were no significantly less than that of the left side（P<0.05）, there were no significant different in TH-positive at right SNp in MSCs groups between 2 weeks and 4 weeks.4. There was no dysembryoma in brain of the PD rats. DISCUSSIONIt is probably safe and effective to transplant MSCs from human cord blood into striatum of PD rats to correct behaviors of Parkinson’s disease rats by the expression of TH.