A Study On Inducting Influence Of Desert Hedgehog On Differentiation Of Neural Progenitor Cells From Embryonic Rat Mesencephalon

The neural precursor cells (NPCs) are self-renewing and multipotent primitive cells that give rise to neurons,astrocytes and oligodendrocytes under certain specified conditions.The exploring of NPCs makes a broad prospect for treatment of neurodegenerative diseases such as Parkinson disease (PD) with cellular transplantation.There are three main strategies which are employed in the cell replacement with neural precursors to treat the neurodegenerative diseases. 1.The neurotrophic or growth factors are administrated to promote the neural precursors in vivo to migrate and differentiate into the specific cell types. 2.The uncommitted neural precursors are transplanted into the specific sites of the brain and differentiate in vivo. The therapeutic effects have been proved however, to be limited because few of engrafted cells can differentiate into the neurons in vivo. 3. Neural precursors are induced to differentiate into the acquired phenotype in vitro, followed by transplantation of these committed neuronal progenitors or immature/mature neurons into the specific region of the brain. The key issue of putting it into effect, however, is to find out a stable inducement pattern to manipulate the conversion of DA neurons in vitro by inducing differentiation of cultured NPCs.The promising findings suggest that the Sertoli cell is an ideal vehicle for providing trophic support to co-cultures from other tissue. But there has been no evidence to show that the precise inducing factor, especially DHH on the development of neural cells. We studied the functional mechanism that Sertoli cells promote the conversion of NPCs in the view of DHH gene, which is expressed by Sertoli cells.The content of present thesis consists of the following 3 parts: Part One: The trophic and promoting function of Sertoli cells on the differentiation of neural precursor cells.Objective To investigate the roles of nutrition and induction of Sertoli cell on cultured NPCs from embryonic mesencephalon in the rat.Methods Sertoli cells from the testis and the NPCs isolated from the ventral mesencephalon in embryonic SD rats (E13.5) were co-cultured. The detection of immunoreactivities of TH was made by immunocytochemistry after 10 days in vitro.Results The numbers of TH-positive cells were 30.56±7.08 and 26.45±6.23 respectively in contact culture and non-contact culture. But the number of TH-positive cells was 1.12±0.06 only in control culture.Conclusion The NPCs from the ventral mesencephalon in embryonic rat can be directly induced to differentiate into the DA neurons by co-cultured with the Sertoli cells.Part Two: Cloning rat DHH gene and detection of its expression in either COS7,NIH/3T3,9L,astrocytes,and Sertoli cells or culture supernatants.Objective To clone DHH gene from rat's testes, reconstruct eukaryotic expression vector, pLXSN/DHH. Infection of DHH into COS7,NIH/3T3,9L,astrocytes and Sertoli cells.Methods1 Cloning DHH cDNA: Total RNA was extracted from rat's testes. PCR production was obtained by RT-PCR method. The fragment cDNA was ligated to pGEM-T Easy vector by T4 DNA ligationase. After being transferred into the DH-5α, white clones were selected and identified by restricted enzymes. The clone with 1220 bp insert fragment was sent to test sequence.2 Constructing pLXSN/DHH: Plasmid pGEM-T Easy/DHH and pLXSN plasmids were extracted, digested with EcoR I and BamH I. The linear fragments were purified and retrieved by glassmilk, then ligated by T4 DNA ligationase. Ligation production was transferred, miniprepared and identified by EcoR I and BamH I. And then large-scale prepared.3 Transfecting DHH into PT67 cells and detection the expression of DHH in COS7,NIH/3T3,9L,astrocytes,and Sertoli cells. When the confluence of PT67 cells was up to 70⁸0%, the complex of pLXSN/DHH with lipofectamine was added. After being selected by G418, PT67 cells which were highly expressed DHH were obtained and identified by immunocytochemistry. The virus supernatant was collected and infected into these cells. We detected the expression of DHH in these cells and cultured supernatants with immunofluorescence, Real-Time PCR and Western Blot.Results1 Cloning DHHcDNA and Constructing pLXSN/DHH: Extracting Total RNA and 28s,18s,5s RNA bands were observed clearly. The 1220bp band was acquired by RT-PCR. After identified by EcoR I and BamH I, the clone with 1220bp insert fragment was further to be identified by restricted enzymes located in vector and fragment. This sequence was proved to be correct compared with that in GeneBank (XM₃43327). The DHH fragment was ligated with linear pLXSN digested by EcoR I and BamH I. After being transferred, mimiprepared and digested by enzymes, the finding of 1220bp insert fragment from pLXSN/DHH vector was reconstructed successfully.2 The expression of DHH in COS7,NIH/3T3,9L,astrocytes, Sertoli cells and supernatants. PT67 cells which expressed DHH highly were obtained and identified by immunocytochemistry. The virus supernatant was collected and infected into COS7,NIH/3T3,9L,astrocytes and Sertoli cells. We could detect the expression of DHH in these cells and culture supernatant with immunofluorescence, Real-Time PCR and Western blot.ConclusionThe eukaryotic expression vector, pLXSN/DHH was constructed successfully. A high expression of DHH can be seen in the COS7,NIH/3T3,9L,astrocytes and Sertoli cells and DHH could be secreted into the culture supernatant.Part Three: DHH induces the differentiation of neural progenitor cells from embryonic mesencephalon of rat into dopaminergic neuronsObjective DHH induces the differentiation of neural progenitor cells from embryonic mesencephalon of rat into dopaminergic neurons Methods We designed 4 groups in the following study. Group 1: The cells ofCOS7,NIH/3T3, and 9L, which all expressed DHH, were contactly and non-contactly co-cultured with the NPCs isolated from the ventral mesencephalon in embryonic SD rats (E13.5), respectively. Group 2: Normal and expressed DHH astrocytes were contactly and non-contactly co-cultured with the NPCs, respectively. Group 3: Sertoli cells overpressed DHH were contactly and non-contactly co-cultured with the NPCs, respectively. And at the same time, blocking antibodies to DHH were adminisstried in another team. Group 4: NPCs were cultured alone. The TH positive cells were detected with immunofluorescence 10 days later.ResultA few TH-positive cells could be detected in the two co-cultral systems at the 10th day in group 1 with no statistic meaning. The numbers of TH positive cells were 24.89±6.32 and 16.57±4.03 in co-cultral systems, contained normal astrocytes contacted and non-contacted respectively with NPCs. The numbers of TH positive cells were 25.39±6.01 and 14.72±2.90 respectively in the systems containg expressed DHH astyocytes and contactly and non-contactly co-cultred with NPCs. In group 3, the TH positive cells number was 30.56±7.08 in cultured NPCs, overexpressed DHH contacted with Sertoli cells, while it was 26.45±6.23 in the non-contact co-cultural system. In the antibody blocking system, the TH positive cells number was 31.36±7.42 (contact co-culture) and 26.07±6.12 (non-contact co-culture), respectively. In group 4, the TH positive cell number was only 1.12±0.06.ConclusionThe protein derived of DHH itself does not seem to have an inductive impact on direct differentiation of NPCs to the dopaminergic neurons in vitro.