| Part one:Objectives:1. To find a method to collect mesenchymal stem cells with a high yield;2. To establish a method on culture and identify mesenchymal stem cells from adipose (ADMSC), bone marrow(BMSC) and umbilical cord from human and swine;3. Select a mesenchymal stem cell suitable for transplantation into Rongchang swine.Methods:Swine ADMSC were isolated from fat tissue with collagenase and induced to differentiation to osteogenic, adipogenic and chondrogrnic cells. The survival curve of the ADMSC at the37℃and38℃were measured using WST-lCell Proliferation Assay Reagent. Swine BMSC were separated to osteogenic and adipogeniccell using density-gradient centrifugalization and the survival curve were delineated with cell counting. Human UMSC were isolated by tissue adherence and the phenotypes were tested by flow cytometer. Swine ADMSC were cultured and the different survival rates were measured under5%or15%fetal bovine serum culture conditions.Result:ADMSC isolated from swine neck fat tissue with collagenase could generate a stable uniform appearance after the second generation. The passage period is five days. They can differentiate to osteogenic, adipogenic or chondrogrnic cells under different culture conditions. Under38℃, they showed a fast growth rate. BMSC isolated from bone marrow of swine femur could generate a stable uniform appearance after the third generation. The passage period was ten days. BMSC could differentiate to osteogenic or adipogenic cells under different culture conditions. UMSC could be isolated from human umbilical cord twenty days of tissue adherence. They showed uniform appearance and array as swirl. The fastest growth rates appeared from the first to the tenth generation which has been passaged every four days. They expressed CD73and CD105, but not CD31and CD34.Conclusion:Human UMSC Isolated and culture facility and they show a fast growth rate and it is suitable for transplantation. Part two:Objectives:To explore the feasibility of stem cell transplantation to inner ear through the subarachnoid cavity in young baby swine.Methods:Dissection of the lumbar and sacral vertebral region in order to measure the size of spinal process and intervertebral space of lumbar vertebral. Methylthioninium chloride was injected into the subarachnoid cavity through the lumbar punctuation. The distribution of the blue dye was examined two hours after the injection. The frozen section of acoustic nerve and temporal bone were examined under laser microscope twenty four hours after injection of DAPI through the subarachnoid cavity.Result:The size of the spinal process of young swine’s lumbar sacral vertebrae was1X1.5X0.2cm which showed an obliquity to the head side. The widest place of the intervertebral space was between L5and S1vertebrae which was about0.5cm. The acoustic nerve was stained by Methylthioninium chloride injected through the subarachnoid cavity. The acoustic nerve could be stained by blue fluorescence after24hours of DAPI injection through the subarachnoid cavity. However, the cochlea sections didn’t have any fluorescence after about30days decalcification treatment.Conclusion:The anatomic structure of young swine’s vertebral column is similar with human. Under condition of well anesthesia, the cerebrospinal fluid could be collected by the lumbar punctuation. Drugs could also be injected through the subarachnoid cavity. This technique can be used to trace cerebrospinal fluid and mark acoustic nerves in the internal acoustic meatus. Part three:Objectives:To examine the distribution of mesenchymal stem cells transplanted through the subarachnoid cavity and the effects on auditory brainstem response (ABR).Methods:The fifth or sixth generations of human umbilical cord mesenchymal stem cells (UMSC) have been transplanted into young congenital deaf albino Rongchang swine through the subarachnoid cavity. Swine with normal hearing were used as controls. ABR were tested before and up to eight weeks after UMSC transplantation. Cochlear samples have been collected at2hrs,3days,1,2,3,4and8weeks after transplantation. Immunohistochemistry has been used to detect the proliferated cell nuclear antigen (PCNA) from frozen tissue of cochlea.Result:The transplanted UMSC have been found at the stria vascularis, the basement membrane and the spiral ganglions of the cochlea3days to4weeks after the transplantation. UMSC cells and their DNA were found also in the cerebrum, cerebellum, medulla oblongata, heart, liver, kidney and lung3days and1week after the transplantation. ABR of the young congenital deaf Rongchang swine displayed an abnormal waveform than before the transplantation.Conclusion:The young Rongchang swine could live well at least8weeks after human UMSC transplantation through the subarachnoid cavity. The UMSC could immigrate into inner ears, central nervous system and periphery organs. The UMSC may improve ABR in the deaf Rongchang swine. Part four:Objective:To explore the clinical features, diagnosis and treatment of the temporal giant cell granuloma (GCG) and to improve the effect of therapy.Methods:Eight cases (4males and4females, age from21to50,4left and4right) of GCG being treated in Chinese PLA General Hospital were retrospectively analyzed from2001to2010, investigate their clinical, imaging and histopathological features, methods of surgery and results of follow-up study.Results:The median disease duration is21months (ranging from5month to60 years). All cases were primarily treated in our hospital except one recurred patient referred from another hospital. The main complains of the patients include hearing loss (5cases), tinnitus (4cases) and local masses (2cases). Imaging examination showed local masses, erosion of temporal bone and skull base. All patients were treated by surgery. Five of them were surgically treated by middle cranial fossa approach and three were treated by combined cranio-auriculo-cevicalapproaches approach. Temporary partial facial paralyzes were seen in three cases and one for cerebral edema after operation. All of these symptoms were healed after treatment. Seven cases have been followed up with mean duration of25.7months (ranging from12to58months). None of them recurred (one lost following-up).Conclusion:It is difficult to diagnose GCG due to no specific clinical and imaging features. The main symptoms of GCG are hearing lose, tinnitus, dizziness, ear pain and local masses. The surgical treatment should be the first choice and the recurrence of GCG was rare. |
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