Transplantation Of GDNF Gene Modified Human Adipose-derived Mesenchymal Stem Cells In A 6-OHDA Mouse Model Of Parkinson's Disease

Part1 Establishment and Assessment of 6-OHDA Induced Mouse Model of Parkinson's DiseaseObjective: To develop a unilateral PD mouse model by injecting 6-hydroxydopamine(6-OHDA)into the striatum.Then assessing the 6-OHDA induced mouse model systemically by evaluating their behavioral performance,immunohistochemical results,dopamine synthetic ability,etc.Methods: Twenty C57BL/6 mice were grouped randomly and equally into PD group and Sham group.PD group received unilateral striatum stereotaxic injections of 6-OHDA,and sham group received equivalent volume saline.Apomorphine(APO)induced rotation and rotarod test were used to assess the motor dysfunction 1-5 weeks after surgery.The striatum protein was harvested from 3 randomly chosen PD mice and tyrosine hydroxylase(TH)content was determined by Western blot.TH immunohistochemical or immunofluorescence staining was performed to estimate the damage degree of the nigra-striatum system.Results:1.The PD group mice began to have dark hair color and decreased free activity in the first week after injection of 6-OHDA,and the hair color and autonomic activity recovered gradually in the third and fourth week after the operation.The weight of the PD group mice decreased in the 1st and 2nd week after surgery,and increased gradually from the third to 5th week.While in the sham group,there was a gradual increase in body weight.In week 4 and 5,the weight of PD group was significantly lower than that of sham group(p<0.05).2.At 5 to 7 days after lesion,APO induced rotation was detected in PD group mice.Eight of nine lesioned mice manifested steady rotating and the overall success rate of PD model was 88.9%.The average tuning number of PD mice was 9.09±1.70 r in 1st week after surgery,which increased significantly in week 2 and 3(11.47±2.20 r,10.79±2.01 r respectively),and decreased gradually in week 4 and 5(7.94±1.19 r,5.12±1.18 r respectively).These results indicate that unilateral striatum injection with 6-OHDA in mice can induce stable acute PD model,and the induced rotation behavior will last for about 5 weeks.3.The latency to fall from the rotating rod of PD group from week 1 to 5 was significantly shorter than that of sham group(p<0.001),which indicates that the motor coordination ability and endurance of PD group mice were greatly affected by the lesion.4.The TH immunohistochemistry and immunofluorescence staining of striatum in PD mice showed that the TH positive nerve fibers of the left(damaged)side were significantly less than the right side,and the ventral part of striatum with remaining innervation was stained.The TH immunofluorescence staining of midbrain showed that TH positive cell bodies in the left side of the substantia nigra(SNc)were significantly less than that in the right side,the loss of TH positive cell bodies in the left side of ventral tegmental area(VTA)was a small amount,TH positive nerve fibers of left midbrain decreased significantly compared with right.5.Western-blot results showed the TH content of left striatum in PD mice was significantly lower than that of the right side(p<0.05).Conclusions: PD mouse model with 6-OHDA lesioned unilateral striatum simulates the progressive pathological progress of PD to a great extent.The motor behavior can be evaluated easily and quantitatively,and lasts long,which means this animal model can beused for further screening drugs or stem cell transplantation research.Part2 Cultivation,Identification of Human Adipose-derived Mesenchymal Stem Cells,Establishment of GDNF Gene Expression Vector,and Cell InfectionObjective: To isolate and cultivate the primary human adipose-derived mesenchymal stem cells(h ADMSCs),and observe the growth and proliferation potency of h ADMSCs.To deliver GDNF gene into h ADMSCs by lentivirus(LV),and construct the GDNF gene expression vector.To identify the h ADMSCs which are modified by GDNF gene.Methods: The primary h ADMSCs were isolated by collagenase digestion,cultured and passaged in vitro.The growth and proliferation of h ADMSCs were observed.The lentivirus carrying GDNF+GFP gene and GFP gene were transferred into the h ADMSCs respectively.Green fluorescence protein(GFP)expression was observed under the inverted fluorescence microscope,and the infection efficiency was calculated.The cell membrane cluster differentiation antigens of h ADMSCs were detected by flow cytometry.Western blot method was used to detect the expression of GDNF and GFP gene in modified GDNF+GFP group and GFP group h ADMSCs respectively.Results:1.The primary cells began to grow adhering to the wall at about 6-12 hours,showing a round or short stick shape.Fifty percent of the cells adhered to the wall in about 24 hours.The cells had a fibroblastic-like morphology of long shuttle in 7th to 8th day,and the cell fusion degree could reach 80% to 90%.The morphology of passage cells was not different from that of the primary cells.HADMSCs can be sub-cultured for 13-14 generations.2.The GFP positive ratio of GDNF+GFP group h ADMSCs was not significantly different from that of GFP group.Cells of the former grew more densely and proliferated faster.3.The results of flow cytometry showed that cultured h ADMSCs were positive for CD73,CD90,CD105,and were negative for CD31,CD34,CD45.4.Western blot results showed there was no significant difference in the expression of GDNF protein among P1,P3 and P6 GDNF+GFP group h ADMSCs.The GFP group h ADMSCs did not express GDNF protein.There was no significant difference in the expression of GFP protein between GDNF+GFP group and GFP group h ADMSCs,and also no difference among P1,P3,and P6 cells.Conclusions: The h ADMSCs infected with LV-GDNF could express GDNF steadily and efficiently in vitro.The h ADMSCs modified by GDNF gene still maintain the characteristics of mesenchymal stem cells,which can be used as the ideal stem cells for transplantation therapy of PD.Part3 GDNF-overexpresing Human Adipose-derived Mesenchymal Stem Cells Treating a 6-OHDA Mouse Model of Parkinson's DiseaseObjective: To study the effect and relevant mechanism of GDNF-overexpressing h ADMSCs transplanting for a unilateral PD mouse model.Methods: First,a unilateral PD mouse model was constructed by injecting 6-OHDA into the left striatum.APO induced rotation was performed on the 7th day after surgery,and 20 PD mice were selected for the h ADMSCs transplantation treatment research.The 20 PD mice were randomly divided into 3 groups,namely GDNF+GFP group,GFP group,and control group(7,7,6 respectively).Another six C57 mice were selected for sham group.The GDNF+GFP group h ADMSCs,GFP group h ADMSCs,and equivalent 1 x PBS were injected respectively into the left striatum of each corresponding group in the 9th day after first surgery,and the sham group was not treated.APO induced rotation was performed on the 14 th,21st,28 th,and 35 th day after first surgery,and the rotarod test was performed on the 5th,12 th,19th,26 th and 33 th day.The mice were sacrificed after the behavioral test.Then,the harvested brain tissues were paraformaldehyde-fixed,sliced,and stained with immunofluorescence.Results:1.The results of APO induced rotation.The average turning number of GDNF+GFP group mice was significantly lesser than that of control group in week 3,4,and 5(p=0.029,0.011 and 0.003 respectively).Compared with GFP group mice,the average turning number of GDNF+GFP group was also significantly lower in week 3,4,and 5(p=0.041,0.022,and 0.012 respectively).It means that APO induced rotation could be alleviated when GDNF-overexpressing h ADMSCs were transplanted into the 6-OHDA lesioned PD mouse model.2.The results of rotarod test.The latency to fall from the rotating rod of GDNF+GFP group mice was significantly longer than that of control group in week 3,4,and 5(p=0.001,p<0.001,p=0.001,respectively).Compared with GFP group,the latency of GDNF+GFP group was significantly increased in week 4 and 5(p=0.011,p=0.031,respectively).The latency of GFP group mice was obviously prolonged compared with that of control group in week 3,4,and 5(p=0.012,p=0.048,p=0.014,respectively).These results indicate that the motor coordination ability of PD mice improved in varying degrees from 2nd to 4th week when transplanted with GDNF-overexpressing h ADMSCs or simple h ADMSCs,and the GDNF+GFP group was better than GFP group.3.GFP immunofluorescence staining showed that h ADMSCs survived in both GDNF+GFP group and GFP group in the 4th week after transplantation,and the number of h ADMSCs in the GDNF+GFP group was significantly higher than that in the GFP group.HADMSCs in the GDNF+GFP group showed a long fusiform distribution in the left striatum,and had a tendency to spread to the surrounding parenchyma.4.The content of TH positive nerve fibers of left striatum in GDNF+GFP group mice was higher than that in GFP group and Control group mice.TH immunofluorescence staining of h ADMSCs was positive in the left striatum of GDNF+GFP group mice.In addition,GFAP staining of h ADMSCs was positive,neural precursor markers Nestin and?-tubulin-III staining were positive,and mature neuron marker Neu N staining was negative.Conclusions: GDNF-overexpressing h ADMSCs transplanting for 6-OHDA lesioned unilateral PD mouse model can effectively improve the motor behavior,and increase the expression of TH in the striatum.GDNF secreted by h ADMSCs facilitates the repair of damaged DAergic neurons and promotes the survival and growth of h ADMSCs itself.HADMSCs transplanted in vivo have the potential to differentiate into DAergic neurons and play a role in cell replacement.