The Inducing Role Of Sertoli Cells And Its DHH Gene On Differentiation Of Neural Precursor Cells From Embryonic Mesencephalon Of Rat In Vitro

It has already known that the neural precursor cells (NPCs) are self-renewing and multipotent primitive cells that give rise to neurons, astrocytes and oligodendrocytes. The exploring of NPCs makes a broad prospect for treatment of neurodegenerative diseases such as Parkinson disease (PD) with cellular transplantation. The key issue of putting it into effect, however, is to find out a stable inducement pattern to manipulating the conversion of DA neurons in vitro by inducing differentiation of cultured NPCs. Currently, there are some problems for this purpose existing in the research field of direct inducement of differentiation of DA progenitors, although quite a few of studies have been done and shown to be promising. These problems include: â‘ There has been no effective method so far for inducing NPCs to differentiate into the specific subtype of neurons; â‘¡The mechanism which controls the differentiation of NPCs is not clear enough to be put into practice; â‘¢Certain inducement factors which are found to have some effectiveness in vitro, have not been verified in vivo. To address these issues, we attempt to explore a new way of producing DA neurons from the cultured NPCs in the present study, i.e. to establish a co-cultural system of the Sertoli cells with NPCs from the mesencephalon of rat fetus, in order to promoting the differentiation of NPCs by Sertoli cells. Meanwhile, 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. Sertoli cells are known to reside in the testis where they provide an appropriate microenvironment for the development of germ cells and stimulate the survival and development of sperm cells. Sertoli cells also secrete a trophic cocktail of growth factors, cytokines and regulatory proteins, a large number of which could be the 'active ingredient'for the conversion of DA neuron. The promising findings suggest that the Sertoli cell is an ideal vehicle for providing trophic support to co-cultures from other tissue. In our study, we found out that the numbers of TH-positive neurons were increased when NPCs co-cultured with Sertoli cells. Desert hedgehog (Dhh) is also known to be a cell-signaling molecule that belongs to the Hedgehog (Hh) secreted protein family, which includes Sonic (Shh), Indian (Ihh), and Desert hedgehog (Dhh). DHHmRNA showed a very restricted distribution, being expressed primarily in the Sertoli cells of developing testes and in Schwann cells in the peripheral nerves. Dhh signaling plays an essential role in the regulation of mammalian spermatogenesis. There are several studies showing that the Shh, a homology of Dhh, can induce the differentiation of motor neurons in the spinal cord and DA neurons in the midbrain. However, there has been no evidence to show that the function of Dhh on the development of neural cells. For these reasons, we cloned the gene of Desert Hedgehog (DHH) from rat, construct its eukaryiotic expression vector and transfect it into COS7 cells. We hope this may be helpful to investigate the function of Dhh on the differentiation of NPCs into DA neurons. The results demonstrated in present study showed that Dhh could promote the differentation of NPCs. The content of present thesis consists of following 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 mesemcephalon in the rat. Methods Sertoli cells from the testis and the NPCs from the ventral mesencephalon in embryonic SD rat were co-cultured. The detections of immunoreactivities of Nestin, β-â…¢tubulin and TH were made by immunocytochemistry after 5,7,10 and 14 days in vitro. Results The number of NPCs with Nestin-positive was decreasing along thetime of the co-culture, while theβ-â…¢tubulin-positive cells and TH-positive cells were increasing. At any stage, the numbers of TH-positive cells were higher in the co-culture, compared with those in the mono-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: DHH gene cloning and its eukaryotic expression vector constructing Objective To clone DHH gene from rat's testes, reconstruct eukaryotic expression vector pLXSN/DHH Methods 1 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 DH5α, 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. 3 Results 4. Extracting Total RNA: 28s, 18s, 5s RNA bands were observed clearly. 5. Acquiring DHH cDNA fragment: About 1220bp band was acquired by RT-PCR. 6. Cloning PCR production into pGEM-T Easy vector and sequencing: 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). 7. Constructing eukaryotic expression plasmid pLXSN/DHH: DHH fragmentwas ligated with linear pLXSN digested by EcoR I and BamH I. After being transferred, mimiprepared and digested by enzyme, the finding of 1220bp insert fragment showed pLXSN/DHH vector was reconstructed successfully. Conclusion Total RNA was got from rats testes, the cloned DHHcDNA was proved correctly, and eukaryotic expression vector pLXSN/DHH was constructed successfully. Part Three: DHH cRNA probes labeling and detecting Objective To label DHH cRNA probes and examine the concentration Methods The sense and anti-sense templates were purified and retrieved by glassmilk after the pGEM-T Easy/DHH was digested by Not I and Nco I restriction enzymes. The cRNA probes were labeled by Dig-cRNA probe labeling kit. Then labeled DHH anti-sense and sense probes were diluted, dotted, rayed by ultraviolet and examined by ALP system. Results The result of dot hybridization was shown that the positive reaction sites were purple-blue, and their densities were well matched with the concentration and dilutions. Compared with the control RNA, it was evaluated that the concentration of anti-sense and sense probes were respectively 150ng/μl and 80 ng/μl. Conclusion DHH probes were labeled, and blot hybridization results showed that the concentration of anti-sense was 150 ng/μl; sense was 80 ng/μl. Part Four: Testing the expression and activity of Dhh protein Objective To investigate whether DHH gene can be expressed and Dhh protein can be secreted out and have effective biological activity or not. Methods 1 Transfecting PT67 cells: When the confluence of PT67 cells is up to 70⁸0%, the complex of pLXSN/DHH with lipofectamine was added. After being selected by G418, the virus supernatant was collected. 2 Transfecting COS7 cells: Adding the virus supernatant of PT67 to the COS7 cells, transfecting for 6 hours. Then collecting the supernatant of COS7 cells.