| Parkinson's disease (PD) is the most common progressively neurodegenerative disorders, its basic clinical symptoms include tremor at rest, rigidity, bradykinesia. postural abnormalities and a freezing phenomenon. It is a progressive and degenerative disease which can lead to lots of movement disorders and its numbers increase, so the social and economic impacts of PD increase over time. The pathological finding in PD is the loss of nigrostriatal dopaminergic (DA) neurones, with a resultant loss of DA and its metabolites DOPAC (3,4-dihydroxyphenylacetic acid). Currently, the main methods for treatments of PD include pharmacological treatment, surgery, cell transplantation and gene therapy. Although Pharmacological treatment can alleviate the symptoms, it can't halt the progression of the disease. Cell replacement is a therapy to replace the loss of dopamine-producing neurons in the substantia nigra by transplantation in order to restore dopamine neurotransmitters; the efficacy is obvious but not lasting. The ideal PD treatment should not only correct the content of insufficientbrain DA, but also protect the remaining DA neurons. The gene therapy is the best way to achieve these goals, becoming the hotspot of PD treatment.Gene therapy is now primarily transferring the target gene to cells and transplanting the cells to brain or directly transferring the purpose gene to brain lesion site by plasmid or virus carrier. Recently, evidence has been provided that bone mesenchymal stem cells (BMSCs) from various sources could overcome the autologous transplantation of immune rejection and ethical features and easy to be cultivated and proliferated in vitro to become an ideal carrier of cells. Commonly used exogenous genes include the biosynthetic enzymes promoting for dopamine synthesis (tyrosine hydroxylase, TH), various neurotrophic factor gene, antiapoptotic factors, antioxidant enzymes gene, etc. TH is a major dopamine synthesizing enzyme, which is the protein logo of DA neurons within neural system. And also found if they put TH gene into striatum of PD model rats, the level of DA in brain increased obviously and the APO (apomorphine) induced rotation improved, which is beneficial to the treatment of PD.Neurturin (NTN) is a growth factor that is structurally and functionally related to GDNF. They belong to GDNF subfamily, which are identified through theirs capacity to nourish, support, protect and damage repair to dopaminergic neurons. NTN has been confirmed to promote survival of embryos midbrain neurons in vitro and vivo, protect mature dopaminergic neurons in different PD model, and prevent neurodegeneration. Overall, it appears that NTN is a very promising treatment of PD.Previous studies found that TH or NTN separately modified cell transplanted into rats can improve the PD rat behavior, restore and increase DA content. But further study found that the combination of two strategies can be more effective than each of them alone, so the current research tends to carry multiple genes. Fan found the TH gene and the aromatic L-amino acid decarboxylase (AADC) gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-OHDA-lesioned rats, compared with rats receiving AAV-TH alone. And now a pIRES vector carrying BMSCs line-derived neurotrophic factors NTN and TH in the treatment of PD has not been seen. This research conceived that these two genes not only could increase the synthesis of DA brain, but also protect the DA neurons to achieve double therapeutic effect. In this study, we firstly separate BMSCs in vitro by density gradient centrifugation and we confirm the cultured and amplified BMSCs on its biological characteristics, proliferation condition and relevant specific antigen expression. Secondly we construct a pIRES vector carrying BMSCs line-derived neurotrophic factors NTN and TH. Then BMSCs are transfected with pIRES-TH-TNT, and the expression of mRNA and protein of NTN and TH are investigated; finally the transfected BMSCs are collected and implanted into the substantia nigra of PD rats. The rotational behavior of rats, the differentiation of transplanted cells, specific neurotransmitters, histology and related genes and proteins were investigated to evaluate the therapeutic effect of the transplanted genes, hoping to find a method of gene therapy for PD.This research was divided into three parts:Part I Cultivation and biological identification of BMSCs of rats in vitroMethods:1. Obtained bone marrow of rats in sterile conditions; separated BMSCs in vitro by density gradient centrifugation for primary culture.2. Subcultured BMSCs and used MTT to measure its growth activity.3. Analyzed the expression of CD29 and CD34 by flow cytometry (FCM) and immune cell chemistry dyeing.Results:1. Within 24 hours after inoculation, the large circular cells began to stick on the wall and grew. After 2-3 days, most cells began stretching. Several days later, cells proliferation regimented exuberant, generally judged from morphologic for BMSCs.2. After subculture, the cells grew rapidly. Cell growth activities showed that the absorbance gradually increased with the passage of time. Cell proliferation was most rapidly from 6 to 11 days,3. Using FCM showed that positive rate of CD34 cell was only 2.40%, while CD29 was as high as 93.93%. Immunocytochemical method demonstrated that the expression of CD29 was positive, CD34 was negative. According to stem cells molecular surface marker molecules, we could determine it was BMSCs. Part II Construction and expression of pIRES-TH-NTN vector in vitroMethods:1. Obtained TH and NTN gene from Pmd18T-TH and pcDNA-NTN, constructed pIRES-TH-NTN vector.2. Using Lipofectamine 2000 transfected BMSCs lines with pIRES-TH-NTN, which was selected under G418.3. The co-expression of TH and NTN mRNA was detected by RT-PCR.4. The protein of NTN and TH was detected by using enzyme-linked immunosorb-ent assay (ELISA) at 24h,36h,48h and 72h, respectively.Results:1. The restriction analysis showed that the two target genes were correctly inserted into pIREs.3. The vatcor pIREs-TH-NTN was successfully transfected into BMSCs, which was detected under G418 selection.4. The expression of NTN and TH mRNA in TH-NTN-BMSCs was higher than that in BMSCs.5. The protein of NTN and TH in TH-NTN-BMSCs was higher than that in BMSCs.Part?Therapeutic effect of TH-NTN modified BMSCs transplantation on PD model ratsMethods:1. PD models were made by two points injecting the 6-OHDA into unilateral striatum of the rats. Rats were randomly divided into two groups:control group and model group.2. After 2,4,6 and 8 weeks, injected APO 0.5 mg/kg into rats' enterocoeliato to test their behavioral.3. Transplanted BMSCs and TH-NTN-BMSCs respectively into right striatum of PD model, rat models were divided into three groups PD group (not transplanted), BMSCs group (transplanted BMSCs) and TH-NTN group (transplanted TH-NTN-BMSCs). Labeled the cells with 5-bromine deoxidizing uracil (5-BrdU) before transplanting,4. After transplanting 2,4,6 and 8 weeks, injected APO to induce behavior and recorded revolution per 30 min.5. Tested DA and its metabolites DOPAC respectively in right and left of PD group and right corpus striatum of other groups using high performance liquid-electrochem-ical measurements (HPLA-EC).6. The expression of TH in PD group and the co-expression of TH and NTN in other groups were detected by using immunohistochemistry.7. Using RT-PCR and Western blotting separately to test TH and NTN mRNA and protein after transplanting.Results:1. Two weeks later, the rats started to appear less dynamic, pilomotorreflex, moving-slowly, bow etc. After APO induced, control group didn't have constant rotation behavior, while model group had rehabilitation side one-way rotating behavior. We continued to record rotating laps of 30min as PD ethology evaluation index. Successful rat models rotating 7 r/min lap reached 84 (73.68%).2. After transplantation, BMSCs and TH-NTN groups could be observed visible independent activities and TH-NTN rats were more obvious. There were statistical differences between TH-NTN group and two other groups in the rotating laps.3. DA and its metabolites DOPAC reduced significantly in right corpus striatum of PD group. DA reduced about 76.30%, and DOPAC reduced 82.79% in 8 weeks.4. Compared with PD and BMSCs groups, DA and its metabolites DOPAC in TH-NTN group increased significantly, with statistical significance5. PD rats were lack of TH positive cells and positive fiber in damaged (right) side, while the contralateral (left) area existed.6. Immunohistochemical results indicated the transplanted cells in TH-NTN group lived longer in the brain compared with BMSCs. Compared with PD and BMSCs groups,the expression of TH and NTN protein detected by using immunohistochemis-try in TH-NTN group increased significantly, which had statistically significant differences.7. The protein and mRNA in TH-NTN rats increased gradually, which had statistically significant differences.Conclusions1. From 2-3 days after separated BMSCs in vitro by density gradient centrifugation, most cells sticked on the wall and began stretching. From 6 to 11 days, cell proliferation was most rapidly. FCM and immune cell chemistry dyeing demonstrated that the expression of CD29 was positive and CD34 was negative, so generally judged from morphologic for BMSCs.2. A vector pIRES-TH-NTN was constructed, then transfected BMSCs lines with pIRES-TH-NTN, the TH-NTN-BMSCs carrying TH and NTN was successfully constructed and the genes TH and NTN transfected into BMSCs could be highly expressed.3. PD models made by two points injecting the 6-OHDA into unilateral striatum are successful. After transplanted TH-NTN-BMSCs into striatum of PD model, the behavior was markedly improved, DA and its metabolites DOPAC increased significantly, and the protein and mRNA in TH-NTN rats increased obviously.4. This study indicated that these two genes not only could increase the synthesis of DA brain, but also protect the DA neurons to achieve double therapeutic effect,which provide experimental basis and new idea for PD gene therapy. |
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