The Research Of Culturing Schwann Cells Of Sciatic Nerve From Adult Mouse In Vitro

Background/Objective:PeriPheral nerve injury has many causes, such as trauma and tumor surgery.The regeneration and function recovery of PeriPheral nerve is one of the hottest issues in the field of neuroscience.The clinical treatment of a severed PeriPheral nerve involves the use of an autologous nerve graft to bridge the gap when it is difficult to suture it directly. But nerve autografting Remains many Problems, i.e. sacrificing one or more functional nerves and donor-site sequelaes include lose of sensation, scarring, etc. Inaddition, the limited availability of donor tissue represents a severe Problem. Inrecent years, more attention has been focused on the development of tissue engineering nerve as substitute for the nerve autograft to avoid these Problems.The PeriPheral nerve tissue engineering technology brings new hope to resolve the difficulties in PeriPheral neuroscience.Peripheral nerve regeneration is a very complicated process,effected from a biological perspective.It is effected by mangy kinds of factors.Peripheral nerve regeneration process,the growth of axonal regeneration, directional and mature from surrounding nerve regeneration is effected by various factors of microenvironment,including cells,extracellular matrix and dispersion factor,there is some degenerated axonal and pith scabbard debris in early regeneration,including cell components such as SC,fibroblasts,mast cells and macrophages,etc.After peripheral nerve damage,Wanllerian degeneration happened in the whole length of the nerve fibres of distal ends due to loss the connection to the body of the neuron with visible axon swelling,irregular appearance,fracture and dissolve changes observed by light microscopy.The near side with the continuity of the body of the neuron exists,and according to different degrees of trauma,one or several several "LangFei node"degeneration was observed.The near side axons of regeneration and grow into the far side is necessary in peripheral nerve regeneration process,as well as the first element of nerve regeneration. And SC is ecactly the most important factors around nerve regeneration microenvironment.SC is PNS peculiar oligodendrocytes, which has a series of special biological characteristics such as hyperplasia, migration and the secretion of various active material, and it is the only peripheral nerve fiber support cells. SC has significant function diversity, in microenvironment for peripheral nerve regeneration.Through the contact between the axons, SC adjust the myelinates, myelination, and participate in the formation of the matrix of peripheral neural stem cells. After Peripheral nerve damage, SC begin to prolifera, and with the blood source macrophages together to clear collapse product, and then produce and secret some material to influence nerve regeneration. SC has an important role in promoting the axonal regeneration in peripheral nerve system. Therefore, the domestic and foreign scholars pay more and more attention to SC and axonal regeneration of the relationship.At present, most studies pay much attention to the changes of the microenvironment of distal ends nerve regeneration after peripheral nerve injury. The distal ends lost the contact to perikaryon after Peripheral nerve damage, the full length of the far side of nerve fibers to the final happened to collapse, called Wallerian degeneration, including degeneration of axons and myelin, leaving basement membrane tube around the axon. SC separate, proliferate to form Bunger belt in the basement membrane tube with a series of changes such as macrophage cell infiltrates, nerve lining collagen deposition product. All of these changes have an extensive touches with SC. So, SC is the main ingredients of microenvironment of nerve regeneration, and is the key factors to promote the regeneration of the axon outgrowth.Once lack of SC in the microenvironment in nerve regeneration, axon or can’t grow, or regeneration rate drops greatly. Therefore,the study on the surrounding nerve regeneration of microenvironment focused on the SC.The number of SC, morphology and biological characteristics of the nerve regeneration change has a direct impact on the environment after peripheral nerve damage. LiYi studied the change of the SC in distal ends of peripheral nerve after injury in adult rats and the results showed that after peripheral nerve injury, SC form change was most obviously in one month; the cell vitality and proliferation capacity is strongest in one week and then gradually become weak. At present, the study for the changes of near side of peripheral nerve injury is less, especially less reports about the change of the number of the SC, morphology and biological characteristics of near side of peripheral nerve after injury. We believe, in peripheral nerve regeneration process, the near side axons of regeneration and grow into the far side, is the necessary conditions of nerve regeneration, is also the first element of nerve regeneration, and SC is exactly the most important factors in nerve regeneration microenvironment.Schwann cell plays the part of seed cell in the process of peripheral nerve repair with tissue engineering technique, and it is the key to build tissue-engineered nerve. So how to get SC tissue-engineered artificial neural needed quickly is very important. There are many research in this respect at home and abroad. SC is a kind of end-stage cells, it is difficult to stick wall and expansion,and it is difficult to get high purity and number of SC with direct draw material to do cell culture. The complex technology, expensive reagents, poor SC purity, restrict the application of artificial nerve in clinic. Many scholars at home and abroad do research to induce stem cells to cells like SC to solve the difficult problem.But induction rate is low, it turned over to stem cells after the induction conditions moved once again and tumorigenicity still limits its further applications.In the actively exploration to improve schwann cells culture condition in vitro, looking for a better get schwann cells method to get a sufficient number of a purer schwann cells in the practice, people found that nerve degeneration is a good method, and the exploration of our experiment is a new method of nerve degeneration in vitro and has unique advantages. The purpose of the nerve degeneration is to create a microenvironment conducive to the nerve regeneration. It is the necessary condition to complete the purpose of the large number of proliferation of schwann cells.Previous studies have shown that SC have different proliferative potential under different developmental stages, and are not functionally and structurally homogeneous. During Waller degeneration of nerve injury, SCs dedifferentiate, express neurotrophic factors, cell adhesion molecules and immature SCs marker glial fibrillary acidic protein (GFAP), and upregulate p75NTR. These may facilitate SC proliferation and attachment, and promote nerve regeneration. In1999, Keilhoff etal. obtained large quantities of SC with high proliferative potentials after in vivo pre-degeneration (Waller degeneration) for a week and in vitro recultivation. In2009, Tomita did the research of nerve degeneration in vitro to evaluate the changes after the degeneration through observing migration degeneration of schwann cells, found that schwann cells in most migration two weeks after degeneration. The degeneration in vitro can provide a large amount of schwann cells with strong proliferation ability and has exciting prospect of clinical application. However, in vivo pre-degeneration requires two surgical operations, is time consuming, and causes delay in treatment. The clinical applications of this approach are also limited due to individual differences and the resulting difficulties in estimating the timing of desired pre-degeneration effect. In our study, sciatic nerves of adult mice were pre-degenerated in vitro in media mimicking in vivo conditions. In2010, Kram et al. have reported in vitro pre-degeneration by incubating sciatic nerves in DMEM with10%FBS for one week and obtained large amount of pure SC.In the peripheral nerve repair process, SC prolified and secret some neurotrophic factors.The degeneration in vivo is a complicated process with variety of cells and factors involved, so we must simulate environment in vivo in order to get the similar result of the degeneration in vitro. We added schwann cells growth need growth factor (forskolin, heregulin-beta1, alkaline fibroblast growth factor) in order to simulate the effect of in vivo and did the corresponding detection.Methods:1. Experimental animals:green fluorescent protein (GFP) transgenic mice. C57mice, male and female are welcome.2. The degeneration in vitro:mice to be put to death by cervical dislocated, cut off and separate the sciatic nerve, put in the DMEM, DMEM+10%FBS, SCCM medium for cultivating and one week later for detection. The degeneration in vivo: mice were in anesthesia by10%pentobarbital intraperitoneal injection, the right sciatic nerve were exposed and cut away from the root of spinal nerve sciatic nerve, and make the cut ends free and1、2、3week later for detection.3. Cell culture and frozen section production:the sciatic nerve tissue in different cultures and fresh sciatic nerve were rinsed, chopped, dissolved with the digestive enzymes, inoculation respectively. SCCM cells were purified and made frozen section after48hour.4. tissue and cellular immune fluorescence detection:take S100β and p75NTR immunofluorescence staining for the frozen section and original generation cells. To observate expression of green fluorescent protein and S100β from tissue slices by laser confocal microscopy and detecting dyeing cells by common fluorescent microscope, computing cells purity with randomly selected five vision.5. taking pictures under Confocal laser observed.6. Statistics processing:the experiment was repeated3times. All count value to±standard deviation mean differences, statistics software SPSS13.0for data analysis. Mean differences between groups compares with the t test, P<0.05suggests a statistically significant difference.Results:1. The GFP mice group:green fluorescent and p75fluorescence expression of section of sciatic nerve tissue was the strongest after one week of the degeneration in vitro,suggest that the best time for schwann cells proliferation is one week after degeneration.2. GFP mice group:green fluorescent expression and p75fluorescence express is the strongest in SCCM group in1,2,3weeks. Compared SCCM with DMEM group, the group DMEM+10%FBS group and control group,schwann cells, the cell purity and cell density have a statistics difference (P<0.05).3. C57mice group:The bipolar and tripolar SCs were all stained positive with S100β immunofluorescence staining for sciatic nerve cells in the cultivation of SCCM medium, the purity was98%with computing random selected three vision. Compared SCCM group with the DMEM+10%FBS group and control group,schwann cells, the cell purity and cell density have a statistics difference (P<0.05).48hours after the original generation cell culture, schwann cells purity of DMEM+10%FBS, SCCM is82.83±3.43%and89.67±3.14%.conclusions: 1. To do the cell culture after the sciatic nerve degeneration in vitro is a good method to culture Schwann cells of adult mice.2. The best time for Schwann cell proliferation is one week after sciatic nerve degeneration.3. The purity of Schwann cells in SCCM is the best compared with DMEM,DMEM+10%FBS and the control group in vivo,and DMEM+10%FBS superior to DMEM.