The Treatment Of Rats With Parkinson's Disease Using Amniotic Epithelial Cells Transplantation And Its Mechanism

Parkinson's disease (PD) is a common degenerative disease of central nervoussystem (CNS) in middle-aged and old people. With the development of the aging ofthe population in human society, this disease has been one of the important diseaseswith a strong impact on human health.The main pathological change of PD is the progressive degeneration and loss ofthe dopaminergic (DA) neurons in the substantia nigra of midbrain, which results incorpus striatum transmitter system function disorder and a series of clinical nervoussystem symptoms, such as static tremor, myotonia, bradykinesia and attitudinal reflexdisturbance. For the present, the principal pharmacotherapy for this disease is tosupplement the DA in corpus striatum, and surgical treatment focuses on the changeof the abnormal activity of neural circuit. Although all these therapies can mitigatethe symptoms of PD to some extent, they cannot prevent the chronic progressive lossof dopaminergic nerons and thus cannot effectively treat PD by the root.Currently, the protective therapy for dopaminergic neurons has been an import-ant trend in the studies on the treatment of PD, and the neural protective measurestaken in the studies include neurotrophic factors, anti-oxidative-stress, anti-apoptosis,excitatory amino acid receptor blocking pharmacon etc. Among them, the uniquedopaminergic neuron protective effects of neurotrophic factors shown in the in-vivoand in-vitro studies on PD, make investigators further perceive the possibi-lity ofneurotrophic factors of becoming a new-generation drug for PD.Since the pathological changes of PD are mainly present in the brain, and thedegeneration of the dopaminergic neurons in substantia nigra is a chronic progressiveprocess, and no neurotrophic factor can pass blood-brain barrier(BBB), theadministration method of long-term, low dose, repeating injection adopted in thestudies using PD animal models at present is difficult to be performed in clinicalpractice. When using neurotrophic factors to treat PD, the ideal administrationmethod is putting the dopaminergic neurons in the environment containing therequired neurotrophic factors, which are released slowly and stably, in order toprevent the degeneration of them. How to overcome the BBB problem and applyneurotrophic factors in the clinical therapy of PD, has been the principal direction inthe research on the neurotrophic factor therapy of PD.In this experiment, immortalized human amniotic membrane epithelial cell strainFL was transplanted into the corpus striatum of the early PD rat model, and theinhibi-ting effects on the degeneration of dopaminergic neurons in substantia nigravia substantia nigra-corpus striatum path were observed by detecting the stablesurvival of transplanted cells and the expression of brain derived neurotrophic factor(BDNF) and neurotrophic factor-3 (NT-3), which had special nutritional andprotective actions on dopaminergic neurons. The objective was to seek an effectivepathway to apply neurotrophic factors in the clinical therapy of PD as soon aspossible. The detailed content of the research was as follows:1 6-OHDA unilateral damage to establish early and medium phase PD ratmodelsIn this study, cerebrum stereotaxic technique was used, and microinjection of6-OHDA into double target spots of unilateral corpus striatum was conducted toestablish the early and medium phase PD rat models simulating the pathologicalchanges in human PD. The experimental results showed: 63.3% of the rat receivingmicroinjections presented obvious rotary motion to the uninjured side (rotation rateexceeded 7 RPM) 4-6 weeks after 6-OHDA was injected into the unilateral corpusstriatum of 60 rats. At the same time, routine method of histology stain, Nissl's bodystain and TH immunohistochemical stain were performed on the histological sectionsof brain tissues. In the uninjured side (left side) of the successfully established ratmodels, there were a great number of substantia nigra dopaminergic neurons ofzonary diagonal arrangement. In the 6-OHDA corpus striatum injured side (right side),the number of substantia nigra dopaminergic neurons decreased, residual TH positivecells could be seen, and the survival rate of the TH positive neurons in the injuredside was 40.3%. This indicated the early and medium phase PD rat models had beensuccessfully established, which provided the basis for the next study on the protectiveactions of transplanted amniotic epithelial cells on the midbrain substantia nigradopaminergic neurons of rats. In addition, the TUNEL fluoroscopic examination onsubstantia nigra and Bax, caspase-3 immunohistochemical stain results showed:There was significant increase in the number of the TUNEL, Bax, Caspase-3 positiveneurons between the uninjured side and the injured side of substantia nigradopaminergic neurons in successfully established PD rat models. This indicated6-OHDA could result in the apoptosis of PD substantia nigra dopaminergic neuronsby inducing the expression of Bax (the positive regulation gene inducing apoptosis) toactivate Caspase-3, which played a key role in the molecular mechanism of apoptosisinduction. As a result, apoptosis is one of the main ways for the decrease of substantianigra neurons in the rats with Parkinson's disease.2 The protective actions of transplanted amniotic membrane epithelial cells onthe substantia nigra dopaminergic neurons in the rats with Parkinson'sdiseaseIn this study, with the combination use of molecular biological techniques,immunohistochemical techniques, and cerebrum stereotaxic techniques, immortalizedhuman amniotic membrane epithelial cell strain FL was transplanted into the corpusstriatum of PD rat models, in order to explore using the neurotrophic factors BDNF,NT-3 from the self-secretion of transplanted cells to prevent the progressive death ofthe dopaminergic neurons in the substantia nigra of PD rats.The results during the study showed amniotic membrane epithelial cells couldexpress BDNF and NT-3mRNA in vitro. The results of immunohistochemistryproved amniotic membrane epithelial cells could secrete neurotrophic factors BDNFand NT-3 by themselves in vivo and in vitro, survive after implanted into the corpusstriatum of PD rat models, and improve the behavioral symptoms of PD model ratswithin 3 months after cell transplantation via the secretion of BDNF and NT-3. Theimprovement was especially obvious in the second week after transplantation. Onlysporadic rotation behavior could be observed in some PD rats transplanted withamniotic membrane epithelial cells during the induction phase, and other behavioraldetection indices were also mitigated. In the 4th week, the behavioral detection resultsslowly returned to abnormal, but there was significant difference between thetreatment group and the control group. The survival status of dopaminergic neurons inthe midbrain injured side of PD rats, was observed through routine method ofhistology stain, Nissl's body stain and TH immunohistochemical stain of substantianigra dopaminergic neurons, and HPLC-ECD was used to assay the content change ofDA and its metabolites in corpus striatum. The statistical results showed: Comparedwith the physiological saline control group and the PD model control group, thesurvival rate of substantia nigra dopaminergic neurons in the amniotic membraneepithelial cell transplantation group were significantly improved 3 months aftertransplantation. The content of DA and its metabolite DOPAC in the corpus striatumin the injured side of the amniotic epithelial cell transplantation group rats wassignificantly higher than that in the physiological saline control group and the PDmodel control group (p<0.01). These results proved the transplanted amniotic mem-brane epithelial cells had nutritional protective actions on the midbrain dopaminergicneurons in PD rats by secreting neurotrophic factors BDNF and NT-3 so as to preventthe progressive death and loss of dopaminergic neurons caused by 6-OHDA andincrease the content of DA in the corpus striatum in the injured side.3 The protective actions of amniotic epithelial cells on the cell damage of PC12cells induced by 6-OHDAIn this study, MTT test, AO in vivo fluorescein label and Fluorescence activatedcell sorter (FACS) detection techniques were used to observe the protective actions ofthe supernatant of the culture fluid of human amniotic membrane epithelial cells onthe cell damage of PC12 cells induced by 6-OHDA. The results showed: After 30μM,40μM and 50μM(final concentration) 6-OHDA had been added for 24h, the vitalityof PC12 cells gradually decreased with the increase of 6-OHDA concentrations;Thenumber of cells with the cell damage manifestations gradually increased, and apo-ptotic change could be seen;There were obvious changes in the cell cycles of PC12cells, appearing as increase of number of cells in G0/G1 phase and decrease ofnumber of cells in S phase. The supernatant of the culture fluid of amniotic epithelialcells could significantly antagonize the toxic actions of 6-OHDA so as to increase thesurvival rate of PC12 cells, reduce the number of the cell damage, improve theinhibiting effects of 6-OHDA on the multiplication of PC12 cells, and prevent celldamage of PC12. These results proved the supernatant of the culture fluid of amnioticepithelial cells have prevented actions on the cell damage of PC12 cells induced by6-OHDA.This study through experiments in vivo and in vitro, demonstrates the effect of thetreatment of rats with Parkinson's Disease using amniotic epithelial cells transplanta-tion. The results provide important theroretical basis for clinical treatment of PD byHAEC.