大鼠角膜缘上皮细胞培养与同体移植的实验研究

目的:观察以明胶为载体培养的角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症的疗效。方法:大鼠角膜缘上皮细胞在铺有明胶载体的细胞培养板上进行培养5d后,角膜上皮细胞移植术前24h用3H胸腺嘧啶核苷标记培养的角膜缘上皮细胞,培养标记的角膜缘上皮细胞对角膜缘干细胞缺乏的大鼠动物模型行角膜缘上皮细胞同体移植术,对移植术后角膜进行观察、病理学检查及同位素检测。结果:大鼠角膜缘上皮细胞可以在明胶载体上培养、增殖、分化为密集角膜上皮细胞层;角膜缘上皮细胞移植术后角膜上皮完整、基质细胞浸润减轻、新生血管减少。病理学检查角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失及基质中炎性细胞浸润减轻。角膜缘上皮细胞移植术后4wk受眼角膜仍可测到3H胸腺嘧啶核苷。结论:角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。

报废熊胆囊同体移植术41例报告

报废熊胆囊同体移植术４１例报告金昌深，金曾范，李泰源，许应天（延边农学院兽医系）自１９８５年国内开始人工引流熊胆汁以来，各家对引流管材料的改进和防治感染进行了大量的研究，取得了一些成绩，但还未能从根本上克服异材引流管的弊病。如胆囊炎、胆囊机化、报废胆...

犬模拟同体移植胆囊的重建术

为解决牛黄培植与狗宝手术后由于胆囊炎而继发的胆囊萎缩、胆囊变性等导致胆囊不能有效地发挥其贮存胆汁与排出的作用,笔者利用内部器官,采用同体组织的移植方法,进行胆囊重建术的试验研究,使免疫排斥现象得以有效降低,大大提高了手术的成功率,延长了移植组织的寿命,保证正常取胆汁,提高了经济效益。现将其试验结果报告如下。

一例骨囊肿行大段同种异体骨移植术的护理

报告一例骨囊肿所致的病理性粉碎性骨折行大段同种异体骨移植术后的护理。密切观察病情,防止术前、术中、术后感染和肢体末梢循环障碍,减少免疫排斥反应的发生,注意术后肢体功能的锻炼是异体骨移植手术成功的关键。

兔角膜缘上皮细胞培养与移植的实验研究

目的观察以明胶为载体培养的角膜缘上皮细胞移植治疗兔角膜缘干细胞缺乏症的疗效。方法切除实验兔角膜缘组织并用NaOH处理制作角膜缘干细胞缺乏动物模型,切除的角膜缘上皮细胞在铺有明胶裁体的细胞培养板上培养5 d后(角膜上皮细胞移植术前24 h)用3H胸腺嘧啶核苷标记,于模型动物上行角膜缘上皮细胞移植术,术后观察角膜修复情况,并做病理学检查及同位素检测。结果 1.细胞培养:兔角膜缘上皮细胞可以在明胶载体上培养,增殖、分化为密集的角膜上皮细胞层。2.移植眼情况:行角膜缘上皮细胞移植术后,模型兔眼角膜上皮完整,新生血管减少;病理检查显示移植眼角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失,基质内炎性细胞浸润减轻;角膜缘上皮细胞移植术后4周移植眼角膜仍可测到3H胸腺嘧啶核苷。结论角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。

Resveratrol prolongs allograft survival after liver transplantation in rats

AIM： To study the immuno-modulatory effect of resveratrol （RES） on allograft rejection after liver transplantation in rats. METHODS： Male Sprague-Dawley （SD） rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into four groups after orthotopic liver transplantation （OLTx）. In the RES A, B, and C groups, RES was given intra-peritoneally once a day （25, 50, and 100 mg/kg, respectively） after OLTx, whereas in the control group, vehicle buffer was given intra-peritoneally once a day. The survival time, serum chemistry, production of cytokines, activation of transcription factor NF-kB, and histopathologic findings were then compared among these groups. RESULTS： The mean survival time after OLTx in the RES C group was significantly longer than that in the control group （16.7＋-1.2 d ,vs9.3＋-0.6 d, P〈0.01）. On the 7th posttransplant day the serum albumin level significantly improved in the RES C group, the serum total bile acid and alanine aminotransferase （ALT） levels were significantly lower in the RES C group, the serum IL-2 and INF-y levels were significantly lower in the RES C group, and the activation of transcription factor NF-kB in peripheral blood T lymphocytes was significantly suppressed in the RES A, B, and C groups in comparison to those in the control group. On the 7^th post-transplant day, a histological examination revealed apparent difference in the severity of rejection between the RES C group and control group. CONCLUSION： RES has an immuno-suppressive property as well as protective effect on hepatocytes under allograft rejection. It might serve as a novel agent for reducing the severity of hepatic allograft rejection in rats.

Massive allograft replacement in management of bone tumors

Objective： To evaluate the functional outcome and complications of allograft replacement in management of bone tumors. Methods： Between March 1992 and September 2002, 164 patients underwent bone tumor resection and massive allograft reconstruction of bone defects. The length of the resected part ranged from 5-35 cm. The resections were classified as marginal or wide resections of the tumor on the basis of the Musculoskeletal Tumor Society staging system. Fresh-frozen allografts were employed as osteoarticular grafts （n = 95）, hemi-condylar （n = 15）, massive （n = 23）, allograft-prosthesis composite （n = 12）, intercalary grafts （n = 15） or hemi-pelvic grafts （n = 4）. Most of the lesions were osteosarcoma and giant cell tumor of bone and located in proximal and distal femur, proximal tibia and humerus. Results： At a median follow-up of 47 months （range, 12 to 168 months） after the operation, 154 of the patients in the study were free of disease and 10 died of disease. Twenty-one （12.8%） patients had local recurrence and 38 （23.2%） nonunion. Late complications included 11 （6.7%） fractures of the allograft and 18 （11.0%） infections of the graft, instability of the joint in the form of subluxation was noted in 13 （7.9%） patients. Ten extremities were amputated due to local recurrence or severe infection. Conclusion： AIIografts can be used for reconstruction of bony defects after tumor resection. AIIograft has nearly similar shape, strength, osteo-inductivity and osteo-conductivity with host bone. AIIograft implantation is a high complication reconstruction method, and the dsk of recurrence increases when less surgical margin achieves.

EXPRESSION OF ICAM-1 AND LFA-1 MOLECULES IN RELATION TO RENAL ALLOGRAFT REJECTION IN RATS

Objective.The purpose of this study was to assess the renal graft expression of ICAM 1(intercellular adhesion molecule 1) and LFA 1(lymphocyte function associated antigen 1)molecule with relation to graft rejection. Methods.Rat kidney transplantation was performed according to the procedure of Kamada with some modification.Experimental rats were divided into 5 groups.The survival time of recipient rats and function of grafts after renal transplantation were observed.The sections of renal graft were stained for monoclonal antibody ICAM 1 and LFA 1, and then quantification of ICAM 1 and LFA 1 expression was accomplished by computer image analysis. Results.ICAM 1 and LFA 1 increased significantly in the renal allograft rejection group as compared with the non rejection groups(P<0 05). Conclusion.Both biopsy of renal graft and monitoring of ICAM 1 and LFA 1 are useful tools in diagnosing and treating acute rejection.

CD28/B7-MEDIATED COSTIMULATION IS REQUIRED FOR PARATHYROID GLAND ALLOGRAFT REJECTION IN RATS

The activation of T cells to differentiate and to proliferate is an essential step in the immune response to antigen, especially in cell mediated acute allograft rejection. Besides the interaction of CD3/TCR complex with Ag/MHC complex presented on antigen presenting cells, a complete T cell activation and proliferation requires a second costimulatory signal. The interaction of CD28/CTLA 4 and B7 is a major costimulatory pathway for T Cell activation. Inhibition of this pathway results in development of antigen specific unresponsiveness and clonal anergy.In present study,the biologic function of anti CD28 monoclonal antibody and its Fab fragment were investigated in vitro and in vivo.The results indicate that mAbCD28 and its Fab fragments could promote the functional recovery of allografts and prolong the graft survival,but could not reverse the acute rejection or induce transplantation tolerance in the rat PTG allograft model. We also found that peripheral TNF α level and NK cell activity were suppressed in the presence of mAbCD28 and its Fab fragments for a relatively long time after PTG transplantation.

Clinical experience with kidney transplantation in patients older than 65 years

Objective:To explore the peculiarities of kidney transplantation in elderly patients and define the perioperative managements. Methods: The clinical data of kidney transplantation in 29 patients older than 65years were reviewed, the eldest being 84 years old and the mean age 68. 1 years. Results: Four episodes of acute rejection (13. 80%) were encountered. FK506 toxicity occurred in one case (3.40%) and lung infection in another (3.40%), who (along with the former 4 patients) all were cured subsequently. In one case, the kidney graft was removed for thrombogenesis of the renal artery. The 1- and 3-year patients/grafts survival of 100% and 96.5% respectively was achieved, with the longest survival exceeding 5 years. Conclusions:Old age was not the absolute contraindication for kidney transplantation. Strict observance of the indications of kidney transplantation and donor selection with well-matched tissue-typing are crucial in elderly patients.Adequate application of immunosuppressants and effective long-term follow-up are also major factors for long-term allograft survival.

Influence of mycophenolate mofetil (MMF) upon thematuration and allo-stimulatory activity of cultured progenitors of dendritic cells and the effectson the tolerance induction in allograft recipients

To investigate the influence of mycophenolate mofetil (MMF) upon the maturation and the allo-stimulatory activity of cultured progenitors of dendritic cells (DCp), and to evaluate the effects of the pre-treated dentritic cells of recipients with MMF on the tolerance induction as well as its possible mechanism, GM-CSF and MMF were added to the in vitro cultured progenitor cells, and the immuno-phenotypical analysis was performed by means of flow cytometry. The secretion of IL-12 was detected by ELISA and the stimulatory activities of DCp on allogeneic T cells were observed by mixed lymphocyte reaction. Twenty-four C57BL/6 mice were divided into 3 groups (each with 8 mice), in which group A of mice accepted allografts of heart from BALB/c mice, group B of mice had received untreated DCp from donors of BALB/c mice 7 days before transplantation, and C57BL/6 mice in group C were treated by injection with MMF-treated allografts of heart from BALB/c mice 7 days before transplantation. The survival times of allografts and the changes of the cytokine levels in sera of the recipient mice were observed after transplantation. The experimental results showed that MMF could significantly inhibit the expressions of the co-stimulatory molecules CD80 and CD86 on DCs and the secretion of IL-12 and the allo-stimulatory activities of DCs were also markedly inhibited. The survival times of allografts in group B of mice were longer than those in group A, while the group C showed the longest survival times of allografts, with a marked reduction in the production of the Th1 type cytokines. It is evident that MMF has a suppressive effect on the maturation and allo-stimulatory activities of the cultured dendritic cell progenitors, thus leading to a donor specific tolerance in heart-transplanted recipients.

The precursor frequency of alloreactive CTLs is proportional to the number of T cell epitope specificities

The aim of this study is to find the experimental evidence that the precursor frequency of alloreactive CTLs is proportional to the number of the T-cell epitope specificities. The number of T-cell epitope specificities was manipulated by pulsing different humor of HLA-A2 restricted peptide（s） onto the T2 cells, which acted as stimulating cells to elicit allo-reaction by co-culturing with peripheral blood lymphocytes （PBLs） of HLA-A2 negative individual. Ten HLA-A2 restricted peptides （all were normal cell components ） were synthesized, and cell peptide extract was prepared by frozen and thawed. T2 cells loaded with different number of peptide（s） were co-cultured with PBLs of an HLA-A2 negative individual; the latter were stained with PKH67 in advance. Then the proliferation was monitored with flow cytometry, and the precursor frequency of the effector cells was ＇analyzed by the ModFit Software. After 6 d of culture, no proliferation was observed in the bulk culture of PBL alone, and obvious proliferation took place when PBLs of the HLA-A2 negative were co-cultured with T2 cells loaded with or without loading peptide（ s）. The precursor frequency of the alloreactive CTLs was 0.052 819 for co-culture with T2 cells loaded without peptide; however it was 0. 030 429 for T2 cells with EBV/LMP2A and 0. 030 528 for T2 cells loaded with a single autogeneic peptide, and increased up to 0. 144 942 for T2 cells loaded with 10 autogeneic peptides; the precursor frequency was 0. 203 649 when co-cultured with T2 cells loaded with miscellaneous peptides extracted from the cytoplasm of T2 cells. This study reveals that the precursor frequency of alloreactive CTLs is proportional to the number of T-cell epitope specificities, and independent of the density of the allogeneic HLA Class Ⅰ molecule. Our findings support the hypothesis that the alloreactive T cell populations comprise miscellaneous T cell clones; each is specific to corresponding pMHC. The novel constellation of peptides presented by allogeneic MHC molecules makes thousands of different epitopes, which account for the exceptional high precursor frequency of alloreactive T cells.

卵巢移植

卵巢移植韩献萍卵巢移植在上世纪末已应用于防治双卵巢切除或绝经期妇女的不良反应，大致到１９３５年，由于自体移植效果不理想，而异体移植效果更差，使得这一技术被长期遗弃。直至近２０余年，因为器官保存、显微外科及移植手术等技术的进步，使卵巢移植这一技术得到了...

Transplantation of corneal stem cells cultured on amniotic membrane for corneal burn: experimental and clinical study

OBJECTIVE: To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns. METHODS: The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns. RESULTS: Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established. CONCLUSIONS: Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

A histomorphometric and molecular study on stress adaptability of freeze-dried bone allograft

Objective To Investigate stress adaptability of freeze-dried bone allograft.Methods Cortical and cancellous allograft were transplanted to each side of the midshaft diaphyseal ulna in two groups of 28 animals.The left transplanted allograft was free from fixation and bore a normal physiological lcad,while the right transplanted allograft was protected from loading by a simple external fixator and bore less load.Animals were sacrificed at the 2nd,4th,8th,16th week after transplantation and specimens were taken out for bone histomorphometry studies and analysis of collagen gene expression by in situ Cdna-Mrna hybridization.Results Labeled surface(LS)and bone mineral apposition rate(MAR)of the normally loaded graft-host bone interface were significantly higher than that of the less loaded side at the 4th,8th,16th week after transplantation.Parameters reflecting the internal repair process of the allograft,such as LS in cortical and cancellous bone or MAR in cortical bone of the normally loaded side were significantly higher than those of the less loaded side at the 16th week after transplantation.The result of in situ hybridization indicated that more osteoblast-like cells expressing the type Ⅰ collagen gene were found in the interface or interior of normally loaded grafts.Conclusion The stimulus of physiologic load can accelerate the early union of allograft-host bone interface and later new bone creep substitution to the necrotic allograft.

Treatment of chronic osteomyelitis with one-stage allograft

Objective： To avoid disadvantages of two-stage cancellus bone autograft, we investigated the feasibility of one-stage allograft for reconstructing the bone defect resulting from debridement of chronic osteomyelitis in limbs. Methods： Between Feb. 1999 and Apr. 2004, 35 cases of chronic osteomyelitis （8 cases of nonunion ） underwent one-stage allograft after debridement in our hospital. Results： Thirty-five cases were followed up for an average period of 28 months （ range, 13 to 55 months）, in which 32 cases （91.43%） were found no infection, and 3 cases （8.57%） were confirmed recurrence of infection. Four out of 8 cases of bone nonunion healed in 9.5 months on average （ range, 3 to 12 months）, and another case also acquired union after redebridement and autograft of iliumdue to infection recurrence 35 days after surgery. Renonunion occurred in 3 cases, 2 out of whom healed after secondary operation with autograft. One case of renonunion and 2 cases of infection recurrence refused further treatment. Conclusions： A high rate of infection arrest can be attained when one-stage allograft is used to reconstruct the bone defect of chronic osteomyelitis after debridement in limbs. Therefore, chronic osteomyelitis should not be regarded as a contraindication to one-stage allogeneic bone grafting. Renonuion, however, achieves a relatively high rate, especially in cases of segmental bone defect.

A preliminary pathological study on human allotransplantation

Objective: To observe the survival of hand allograft under the state of immunosuppression and the pathological changes of rejection in the recovery process. Methods: The biopsies of the skin, nerve, muscle, tendon and bone tissue of hand allografts during different stages from 1 day to 7 months after operation were observed using routine histological technique. Results: No significant changes due to rejection in skin, nerve, muscle and bone tissue were observed. But different degrees of weak rejective changes were found on the wall of blood vessels; in the muscle and nerve the reactions were markedly stronger than those found in skin tissues. Conclusions: The rejection in deep tissues should be monitored in controlling the rejection of hand allograft.