| PrefaceAs one of the common endocrine diseases, DM can lead to kinds of complications threatening health of patients. Building a endogenous insulin - secretion system is able to cure the DM permanently. Nowadays only pancreas or islets transplant fulfills the purpose. The islet transplant is the cellular transplant, which has the advantages including; safer, simpler, more convenient for the modifying in vitro, lower rate of side - effect and easier to repeat than the pancreas transplant. Although dependents the extrogenic insulin more than the islet transplant has become, the islet transplant is a prospective method.Nevertheless, there are still unsettled problems in the islet transplant. First, how to get the high - quality islet? At present, the isolation of islet has many methods which include routine isolation, collagenase digesting and auto isolation system, et al. From experimental aspect, collagenase digesting and Fi-coll grading centrifugal purifying islet are ideal methods. Second, how to minimizing the rejection which is the key in the long - term islet transplant? Because after transplantation, islet is more easily attacked by the immune system and loses its function than pancreas. The commonly used non - specific immuno - suppression protocol can surely expand the life span of the graft but with the higher possibility of infection and neoplasia.MSCs (Mesenchymal stem cells) is a kind of non - hematogenous stem cell in the bone marrow, which can be induced to differentiation of sorts of tissue cells with special immune characteristics. In one hand, MSCs won't stimulate the immune reaction for its little immunogenicity to avoid the attack of cytotoxic T cell and NK cell; in the other hand, it could regulate the immune system by lower down the reactivity of body immune system and prohibit the proliferation of T cell. All of above suggests that the MSCs can do a lot in the transplant.In addition, the effective inhibition of apoptosis and promotion of cell living will be helpful in the long - term functioning of transplantation. AKT, also called protein kinase B, is a member in the family of Serine/Threonine prokinase. AKT's molecular weigh is 57KD, and plays a key role in the cell living, proliferation, metabolism and apoptosis. AKT is divided into 3 sub -types, namely AKT1, AKT2, AKT3, having almost 82% congenitics. AKT1 exists widely among kinds of tissue and can be activation by many activator; AKT2 exists widely too and is overly produced in the cells of ovary and pancreas carcinoma; and AKT3 mainly comes from the brain and testis. Many unknown fields still existing in the function mechenism, however, AKT, as an important component of living singal, will definitely promote the basic and clinical application with further study.Our trial discusses the new method for the long - term survival of islet by treating rat DM using simultaneous MSCs modified with AKT1 and islet transplant according to the characteristics of AKT and MSCs.MethodAnimal: Male Wistar rat of 1 month for MSCs procurement, Wistar rat of either sex with weigh ranging from 250 - 300g as the donor and male SD rat with weigh ranging from 200±5g as receipient. IDMM rat model made by intraab-dominal injection of 2% STZ( resolved in citrate buffer with PH 4.5, dosage is 60mg/kg) 14 days ahead of transplant. The rats having the blood glucose higher than 16. 7mmol/L for three continuous days are defenite by the DM rats which are randomly divided into 4 group: DM control 6, simple islet transplant 6, simultaneous stem cell and islet transplant 12, simultaneous AKT1 modified with MSCs and islet transplant 18.Islet isolation: Retrograde perfusion of collagenase solution(prepared with cold Hank's solution, dosage is 1. 5mg/ml) 10 - 12ml into the rat pancreas duct,fully inflate the pancreas. Digestion by vibration in the water bath of 37± 1℃. 1mol/L NaOH is occationally added into the bath keeping the PH at 7. 8 1. At the end of digestion, double centrifuge and washing followed by screen by 20. Purifying islet by Ficoll graded centrifuge at concentration of 25% , 23% , 20.5% and 11%. Aspirate the 11% and 20.5% Ficoll solution containing islet, ready for use after washing by RPMI - 1640. DTZ stain is used to decide the purity of the isolated islet. The amount of islet cell = DTZ positive cell group of three samples 3 20 total amount of sample (ml). AO/EB fluro - stain showed the viability of cell and the insulin secretion trial by the ex vivo glucose stimulation showed the islet activeness.MSCs cultrue: Culture uesd the whole blood wall adhesion, bilateral femoral, tibia and humerus were taken from the one month rat. Cells in the bone marrow cavity were washed under aseptic condition then blow into suspended fluid. Then planted in the 25cm2 culture bottle. Culturing in the 5%CO2 satuated wet worm box of 37℃, record the growth status of MSCs by described the growth curve of generation the firsth, third and fifth. Generation P3 of MSCs was selected by its surface marker with the FCM triple antibody (CD34,CD44,CD90).AKT1 construction and modification: 300mg rat liver tissue procured for the RNA isolation. The first cDNA strain was produced by reverse transcript after the RNA quality tested and followed by PCR of AKT1 gene. After constructing the expressiong carrier of nuclea and extracting large amount of apo carrier pcD-NA3 - AKT1, the enzyme incision identification were finished. Modifying the MSCs in the log growth period, the MSCs modified with AKT1 were tested with RT - PCR and Western blot.Subcapsule transplant: Aspirating the transplanted material into the micro sampler, the islet and/or MSCs (modified or non - modified) were concentrated in the end after centrifuge. The DM recepient rat was of anesthesia with ethyl ether, then lying on the back and disinfecting the operation area, expose the right kidney by sublabial oblique incision, pulling out the kidney and inject the islet into the kidney subcapsule, and at last putting the injected kidney back into ab-domial cavity and close the incision with disinfection again.Simple islet transplant: inject 300IEQ islet.Simultaneous MSCs and islet transplant: the amount of MSCs in the group of AKT1 modified or non - modified was about 1×106 on average, transplant -ing with 300IEQ islet simulataneously respectively. Controlled DM rat: Injection the same amount of 1640 solution ( about 15 -20ml). Blood glucose monitor; Blood glucose was tested by the potal blood glucose tester. The blood glucose was tested every day after transplant for 10 days and twice a week thereafter, 6 rats were randomly selected for blood glucose test every time.Insulin test: The insulin was tested on the fourth day and eleventh day post-operation after simultaneous MSCs and islet transplant, and once a week thereafter, with blood drain from the intraorbit vein for 1ml each time, lying stable and centrifuge after serum suspended sent to test.OGTT: Fasting 12hr - 14hr before trial and feeding into the stomach at 2g/ kg with 50% glucose, the blood glucose of 0, 15, 30, 60, 90 and 120 min were tested, and the trial was put on at the third day after normalization of blood glucose, 2 weeks and 4 weeks after transplant.Histomorphology test; 6 rats were killed in each group at the 10th day after transplant; 6 rats from transinfected and non - transinfected group were killed; 6 transinfected rats were killed 3 month after transplant and observing the kidney' s morphology change and islet by HE and insulin - 6 immunohistochemical stain.Statasticis analysis: all the data expressed in mean±SD. T test was used and P <0.05 was considered having statitical significance.Result1. 600IEQ islet could be extracted from every rat, the DTZ stain shows purity more than 90% and islet viability more than 98%. the ex vivo glucose stimulation test showed the average amount of insulin in the hypoglycemia solution is80.7±6.3μIU/ml , the average amount of insulin in the hyperglycemia is257. 4±12μIU/ml, the Secretion Index (SI) is3.64±1.92.2. The growth curve of rat generation the first, third and fifth cell were unanimous, the trend were on accord, the uniformity of MSCs surface marker with the triple fluroantibody was about 95%. Positive CD44, CD90 cells amounted to 95.12% , and the cells of CD90 positive with negtive but CD34 was amount to 93.64%.3. successful AKT1 expression apo carrier was confirmed by the BamHI and EcoRI bi - enzyme electrophoresis and sequencing plasmids, MSCs, was modified by via Lipofectamine 2000, the result of RT - PCR and Western blot showed the AKT1 gene, mRNA and protein expression in the MSCs.4. blood glucose monitor, insulin level and OGTT: The blood glucose begin to decrease at the first day posttransplant, and normalization of blood glucose was achieved at the third after transplant. (1) simple islet transplant group: the survial time of islet was 6 days around, OGTT after normalization of blood glucose showed no stastical difference with normal rat. No insulin test applied for the limitation on the blood sample amount. (2) simultaneous stem cell and islet transplant group: the normalization of blood glucose could be kept for 25 days and the insulin and blood glucose secretion were on accord, the first OGTT had not any stastical significancy compared with normal rat and the second OGTT showed the trend of decreasing. (3) simultaneous AKT modifying MSCs and islet transplant group: blood glucose, insulin level and all three time OGTT had no stastical difference with the normal group.Histomorphology test: (1) first sample: the rejection of simple transplant was obvious, the insulin secretion of islet was scare, while the other two group showed more insulin secretion. (2) second sample; stem cell group had rejection and decreas in the secretion of insulin while the transinfected group had no changes. (3) thired sample:transinfected still showed no change at all.Conclusion(1) collagenase in situ perfusion and Ficoll grading centrifuge can extract high quality islet.(2) kidney subcapsul is a suitable site for the islet transplant.(3) MSCs can prolong the survival time of islet.(4) AKT can protect the survival of islet.
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