| BackgroundPain is a common clinical symtoms, and neuropathic pain itself has already been considered as a disease that could impair the quality of life of the patients. The treatment of chronic pain with traditional drugs has many unresolvable side effects and complications, especially the tolerance and dependence for long-period drug delivery that could cause the ineffectiveness of the treatment. An important issue of pain research is to find a novel method of analgesia that is safe and effective. Gene therapy to alleviate pain might avoid some drawbacks of classical drugs. Moreover, the gene-transfer techniques might improve the effect of present therapies or provide novel ones. Preproenkephalin gene is one of three genes encoding endogenous opioid peptides. Its offspring, enkephalin, is synthesized in a wide variety of central and peripheral neurons, including substantia gelatinosa intemeurons of the dorsal horn and chromaffin cells of the adrenal medulla. Enkephalin is an important mediator in the endogenous analgesic system of central nevous system. Neural progenitor cell (NPC) is the kind of cells with the capacity of proliferation, self-renewal and multi-differentiation and could be the source of neural cells. Many researches have demonstrated that NPC may have the clinical potential for the therapies of cell transplantation and gene transfer. Immortalized neural progenitor cell, which also possess the capacity of self-renewal and could proliferate infinitely, can produce a large number of neural progenitor cells with stable phenotype and genotype. In conclusion, immortalized neural progenitor cell is the ideal cell vehicle for gene transfer to central nervous system. The aims of this study are to construct the immortalized rat neural progenitor cell strain genetically modified by human preproenkephalin gene, and then transplant the cell into rats with chronic neuropathic pain subarachnoidly in attempting to evaluate its analgesic effects. This work may provide a novel technology and method for chronic pain therapy.Methods and Results1. The construction of immortalized rat neural progenitor cell strain induced by SV40 large T antigen geneMethods Neural progenitor cell (NPC) from subependymal zone of postnatal 24 hours SD rat were isolated and cultured adherently with serum-free neurobasal medium containing B27, epidermal growth factor and basal fibroblast growth factor. Plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigen gene (SV40Tag) were transfected into primary cultured NPC using lipofectin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Anti-nestin antibodies were used to identify the cultured cells. The specific molecular marker of neurons and astrocytes were detected using Immunocytochemistry method to investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cells was identified by RT-PCR, southern blot and Immunocytochemistry method.Results Primary cultured cells were nestin positive cells and also stained positive for BrdU. After being induced by fatal bovine serum, they could differentiate into three principal cell types in CNS, as defined by cell-type-specific markers for neurons, astrocytes, or oligodendrocytes. After gene transfection and puromycin selection, One anti-puromycin cell clone was obtained, which was microtubule-associated protein-2 positive cell with the capability of proliferation and could differentiate into microtubule-associated protein-2 or glial fibrillary acidic protein positive cells. The existence of SV40Tag cDNA and the expression of mRNA and protein of SV40Tag were confirmed in transfected cells. The transfected cells were expanded to immortalized cell strain maintained for more than 50 passages, named as immortalized neural progenitor cell (INPC). 2. Study of biological characteristics of immortalized rat neural progenitor cellsMethods The continuously passaged immortalized rat neural progenitor cell strain (INPC) and primary cultured NPC were adopted. Morphology and growth features after subculture, freezing and recovering were observed and compared. Ultrastructure of cells was observed by transmission electron microscope. The cell proliferation rate and cell cycle were detected by bromodeoxyuridine labelling and flow cytometry. The experiment of nude mice transplantation was also used to investigate the tumorigenesis of INPC. The expression of MHC I and II molecular were both detected before and after the differentiation of INPC to evaluate its immunogenisis.Results INPC were elliptical or triangular cells with two or three short axons and still remained monolayer, anchorage dependent and attachment-inhibited growth. Subculture, freezing and recovering had no influence on cellular shape and proliferation of INPC, which has been maintained for more than 50 passages. Compared with NPC, proliferation capability of INPC was stronger, its proliferation index and proliferation rate were increased, population doubling time was shortened. No neoplasm was found in the experiment of nude mice transplantation. Under the normal cultured condition, few INPC cells expressed MHC I and II molecular. After differentiation of INPC induced by 5% fetal bovine serum, the expression of MHC I and II molecular could not be detected.3. The construction of immortalized rat neural progenitor cell strain genetically modified with human preproenkephalin geneMethods Recombinant adeno-associated virus 2 type vectors containing hPPE or enhanced green fluorescent protein (eGFP) were transferred into INPC respectively. The expression of green fluorescent protein was observed after transfection and the efficiency of transfection was measured. Anti-nestin antibodies and SV40Tag antibodies were used to identify the transfected cells. The specific molecular marker of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells after being induced by 5% fetal bovine serum. The expression of hPPE in the transfected cells and the levels of Leu-enkephalin in culture medium were determined with immunocytochemistry, RT-PCR and radioimmunoassay.Results EGFP expression was detected as early as 12h after transfection. The number of eGFP positive cells reached about 90% after 72h. The INPC transferred with hPPE were confirmed as nestin positive and SV40Tag positive cells and could differentiated into neuron and astrocyte by the induction of fetal bovine serum. The expression of hPPE and the levels of Leu-enkephalin in culture medium in INPC transferred with hPPE were higher than in the INPC without transfection (P<0.01).4. Analgesic effect of intrathecal transplantation of immortalized rat neural progenitor cells genetically modified with human preproenkephalin gene on chronic neuropathic pain in ratsMethods 40 adult female Sprague-Dawley rats (150～200g) were randomly divided into five groups: Naive group, Sham group, SNI group, INPC group and INPC/hPPE group. INPC or INPC genetically modified with human preproenkephalin gene (INPC/hPPE) pre-incubated with Bromodeoxyuridine in vitro were transplanted in the subarachnoid space of lumbar 4 to 6 of rats in INPC group and INPC/hPPE group one week after right side spared nerve injury (SNI) respectively. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of all animals were tested once a week from one week before SNI to eight weeks after cell transplantation. The spinal cord of L4-6 was removed eight weeks after cell transplantation, sliced and examined for BrdU and Leu-enkephalin expression using immunohistochemistry, then the expression of hPPE was assessed in the dorsal horn, the content of Leu-enkephalin in the spinal cord was measured by radioimmunoassay.Results Allodynia-like behaviour and the decrease of MWT and TWL levels were observed one week after SNI. The tactile allodynia induced by SNI was significantly alleviated and MWT and TWL return to the normal levels in INPC/hPPE group at the third week after cell transplantantion. The grafts in the surface of dorsal horn were still stained positively for BrdU eight weeks after transplantation. The expression of hPPE and the level of L-EK were higher in INPC/hPPE group than other groups (P<0.05). 5. Statistical AnalysisAll of the analyses were performed by SPSS 10.0 software package. Measurement data were presented as means ± SD. The differences between multiple individual means were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between multiple individual rates were analyzed by Chi-square test. P < 0.05 was considered statistically significant in all tests.ConclusionImmortalized neural progenitor cell strain was established successfully in our study. The INPC cells retain the biological characteristics of its original cells and belong to benign transformed cells. They could be used safely as vehicle in transgenic cellular implantation for pain therapy. Immortalized rat neural progenitor cell modified with human preproenkephalin gene was subsequently established. After being transplanted into rat with chronic neuropathic pain intrathecally, INPC with hPPE could expess Leu-enephalin long-periodly and alleviate the hyperalgesia continuously. Our study provides a long-term analgesia method, which is safe and effective.SummaryAn immortalized neural progenitor cell strain was established successfully with gene-transfer techniques mediated by liposome in our study. The biological characteristics of the cells were well studied. The establishment of this cell strain could solve the problem of quantity shortage and limited lifetime of primary cultured cells, and provides a safety and reliable cell platform for the treatment of central nervous system diseases and transgeneic cell transplantation. The techniques of establishing immortalized cells shows great prospect in clinical application. The transplantation of immortalized neural progenitor cells modified with analgesic gene is an effective and safety cell analgesic method with great feasibility. It not only be a pilot-study for the therapy of intractable pain, but also may establish the foundation for the clinical application of gene therapy.
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