我国器官移植犯罪刑事政策初探

现行器官移植刑事立法的合理之处表现在犯罪圈的适度扩大,刑罚配置的合理化,与器官移植法衔接的一体化,刑事政策的宽严相济化。但其也存在设置的功利性、仓促性和应急性、刑事政策的单一化、立法的协调化不够和刑事法网编制不严密的不足。我国器官移植刑事政策需通过强化刑法解释、拓展犯罪种类以及增进刑法与器官移植法律法规的"无缝对接"等方式不断调整。

中国《人体器官移植条例》评析

人体器官移植是医学科学的重大进步,已经成为拯救器官功能衰竭病人的重要手段。该文采用比较方法分析了中国第一部《人体器官移植条例》存在的不足,并就完善中国人体器官移植条例从可移植器官范围、基本原则、捐献人范围和脑死亡等方面提出了建议。

Anti-CD132 Monoclonal Antibodies Inducing T Cells Apoptosis after Alloantigen Stimulation and Its Possible Clinical Applications

Aim To investigate the mechanism of anti-CD132 monoclonal antibodies (mAbs)inhibiting T cells proliferation in vitro, and their potential values for clinical use. MethodsBALB/c and C57BL/6 mice splenocytes were harvested for two-ways mixed lymphocyte culture (MLC).Anti-CD132 mAbs (final concentration 100 mg·L^(-1)) were added in MLC on day 0 (group 1) or day 3(group 2). Fluorescence activated cell sorting (FACS) was used to measure the proliferation(carboxy-fluorescein dia cetate, succinimidyl ester, CFSE), apoptosis of T cells (PE-CD3,FTTC-annexin-v), and cell cycle (pro-pidium iodide stain) . The expression of survivin in T cellswas detected by immunochemical stai-ning. Re-sults Multi-generation CFSE-labeled splenocytes werefound dividing and their fluorescent strength decreased in MLC. There was no noticeable change influorescent intensity in group 1 and group 2. On day 3, apoptosis induced by anti-CD132 mAbs wasdetected in part of T cells, but was not detected in the former two days in group 1. In group 2, thenumber of cells in M phase (activated T cells) decreased and apoptot-ic cells increased on day 4.The phenomena were not observed in control group (P < 0.01). Expression of survivin in T cells wasdetected in control group but not in groups 1 and 2. Conclusion Blockade of CD132 signaling pathwayinhibits T cell proliferation in vitro by means of inducing activated alloreactive T cell apoptosisbut not the resting T cells. Anti-CD132 mAbs may be candidates for clinical applications.

台大医院一次同步完成六个器官的移植

台大医院最近一次完成心、肺、肝、肾等6个器官的移植,创下台湾地区难得一见的记录。 这项长达17个小时的马拉松手术从1995年12月9日下午6时开始,于10日上午11时20分完成。6个器官都来自同一个捐赠者,在摘取器官以后,分别由各领域医师进行移植,共动用了近100名医护人员。移植后的器官皆已发挥预期的功能。

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

Effects of mycophenolate mofetil vs cyclosporine administration on graft survival and function after islet allotransplantation in diabetic rats

AIM: To develop an experimental model of islet allotransplantation in diabetic rats and to determine the positive or adverse effects of MMF as a single agent. METHODS: Thirty-six male Wistar rats and 18 male Lewis rats were used as recipients and donors respectively. Diabetes was induced by the use of streptozotocin (60 mg/kg) intraperitoneally. Unpurified islets were isolated using the collagenase digestion technique and transplanted into the splenic parenchyma. The recipients were randomly assigned to one of the following three groups: group A (control group) had no immunosuppression; group B received cyclosporine (CsA) (5 mg/kg); group C receivedmycophenolate mofetil (MMF) (20 mg/kg). The animalswere killed on the 12th d. Blood and grafted tissues were obtained for laboratory and histological assessment. RESULTS: Median allograft survival was significantly higher in the two therapy groups than that in the controls (10 and 12 d for CsA and MMF respectively vs 0 d for the control group, P＜0.01). No difference in allograft survival between the CsA and MMF groups was found. However,MMF had less renal and hepatic toxicity and allowed weight gain.CONCLUSION: Monotherapy with MMF for immunosu ppression was safe in an experimental model of islet allotransplantation and was equally effective with cyclosporine, with less toxicity.

Tissue-engineered graft constructed by self-derived cells and heterogeneous acellular matrix

Background： Endothelial and smooth muscle cells were used as seeding cells and heterogeneous acellularized matrix was used as scaffold to construct the tissue-engineered graft. Methods： A 2 weeks piglet was selected as a donor of seeding cells. Two-centimetre length of common carotid artery was dissected. Endothelial cells and smooth muscle cells were harvested by trypsin and collagenase digestion respectively. The isolated cells were cultured and expanded using routine cell culture technique. An adult sheep was used as a donor of acellularized matrix. The thoracic aorta was harvested and processed by a multi-step decellularizing technique to remove the original cells and preserve the elastic and collagen fibers. The cultured smooth muscle cells and endothelial cells were then seeded to the acellularized matrix and incubated in vitro for another 2 weeks. The cell seeded graft was then transplanted to the cell-donated piglet to substitute part of the native pulmonary artery. Results： The cultured cells from piglet were characterized as endothelial cells by the presence of specific antigens vWF and CD31, and smooth muscle cells by the presence of specific antigen a-actin on the cell surface respectively with immunohistochemical technique. After decellularizing processing for the thoracic aorta from sheep, all the cellular components were extracted and elastic and collagen fibers kept their original morphology and structure. The maximal load of acellular matrix was decreased and 20% lower than that of untreated thoracic aorta, but the maximal tensions between them were not different statistically and they had similar load-tension curves. Three months after transplantation, the animal was sacrificed and the graft was removed for observation. The results showed that the inner surfaces of the graft were smooth, without thrombosis and calcification. Under microscopy, a great number of growing cells could be seen and elastic and collagen fibers were abundant. Conclusion： Cultured self-derived endothelial and smooth muscle cells could be used as seeding cells and heterogeneous acellularized matrix could be used as scaffold in constructing tissue-engineered graft.

Structural basis of immunosuppression by the therapeutic antibody daclizumab

Interleukin-2 （IL）-2 signaling plays a pivotal role in the activation of immune responses, and drugs that block this pathway have been shown to be effective for the immunosuppression in patients with organ transplantation to alleviate/eliminate allograft rejection. The first humanized monoclonal antibody （mAb） daclizumab falls into this category and shows high specificity and affinity against a key component of the IL-2 receptor complex, namely IL-2Ra. To reveal the molecular mechanism of the inhibition of the IL-2 signaling pathway by dacllzumab, we determined the crystal structures of the daclizumab Fab in free form and in complex with the IL-2Ra ectodomain at 2.6 and 2.8 A resolution, respectively. The daclizumab Fab adopts a similar conformation in the presence or absence of the IL- 2Ra ectodomain. The antigen-binding site of daclizumab is mainly composed of live complementarity determining regions （CDRs） that form a large positively charged surface depression and two flanking patches that are generally hydrophobic. The conformational epitope consists of several discontinuous segments of the IL-2Ru ectodomain, a large portion of which overlaps with the regions that interact with IL-2, suggesting that the binding of daclizumab to IL-2Ra would prevent the IL-2 binding to IL-2Ra and the subsequent formation of the IL-2fIL-2Ra[~/c complex, and therefore block the IL-2 signaling pathway. These results also have implications for the design and development of improved mAb drugs targeting IL-2Ra.

Beyond tumorigenesis: cancer stem cells in metastasis

The importance of cancer stem cells （CSCs） in tumor-initiation has been firmly established in leukemia and recently reported for a variety of solid tumors. However, the role of CSCs in multistage cancer progression, particularly with respect to metastasis, has not been well-defined. Cancer metastasis requires the seeding and successful colonization of specialized CSCs at distant organs. The biology of normal stem cells and CSCs share remarkable similarities and may have important implications when applied to the study of cancer metastasis. Furthermore, overlapping sets of molecules and pathways have recently been identified to regulate both stem cell migration and cancer metastasis. These molecules constitute a complex network of cellular interactions that facilitate both the initiation of the pre-metastasis niche by the primary tumor and the formation of a nurturing organ microenvironment for migrating CSCs. In this review, we surveyed the recent advances in this dynamic field and propose a unified model of cancer progression in which CSCs assume a central role in both tumorigenesis and metastasis. Better understanding of CSCs as a fundamental component of the metastatic cascade will lead to novel therapeutic strategies against metastatic cancer.

How regenerative medicine and tissue engineering may complement the available armamentarium in gastroenterology?

The increasing shortage of donors and the adverse effects of immunosuppression have restricted the impact of solid organ transplantation.Despite the initial promising developments in xenotransplantation,roadblocks still need to be overcome and this form of organ support remains a long way from clinical practice.While hepatocyte transplantation may be effectively correct metabolic defects,it is far less effective in restoring liver function than liver transplantation.Tissue engineering,using extracellular matrix scaffolds with an intact but decellularized vascular network that is repopulated with autologous or allogeneic stem cells and/or adult cells,holds great promise for the treatment of failure of organs within gastrointestinal tract,such as endstage liver disease,pancreatic insufficiency,bowel failure and type 1 diabetes.Particularly in the liver field,where there is a significant mortality of patients awaiting transplant,human bioengineering may offer a source of readily available organs for transplantation.The use of autologous cells will mitigate the need for long term immunosuppression thus removing a major hurdle in transplantation.

Intrathymic inoculation of donor liver specific antigen alleviates rejection of liver allotransplantation

Use and effects of liver specific antigen in orthotopic liver transplantations were researched in this study. Group I:syngeneic control (Wistar to Wistar); Group II:acute rejection (SD to Wistar ); Group III: Thymic inoculation of SD rat LSA day 7 before transplantation. The observation of common situation and survival time, rejection grades, NF κB activity of splenocytes and IL 2mRNA expression of grafted liver were used to analyze acute rejection severity and immune state of animals in different groups. The common situation of group I was very well after transplantation and no signs of rejection were found. Recipients of group II lost body weight progressively. All dead within day 9 to day 13 posttransplantation; median survival time was 10.7 ±0.51 days. It was an optimal acute rejection control. As for group III, 5 out of 6 recipients survived for a long time and common situation was remarkably better than that of group II. Its rejection grades were significantly lower than that of group II( P <0.05). NF κB activity was only detected in group I at day 5 and day 7 after transplantation, whereas high activity of NF κB was detected at all time points in groupII and the low NF κB activity detected in group III was significantly lower than that of group II ( P <0.05). No IL 2mRNA expression was detected at any time point in group I,whereas high level expression was detected at all time points in group II and the low level expression only detected at day 3 in group III was significantly lower than that of group II ( P <0.05). Conclusion: LSA is an important transplantation antigen which is involved directly in the immunorejection of liver transplantation. We report here for the first time that intrathymic inoculation of LSA can alleviate the rejection of liver allotransplantation; and that grafts can survive for a long time thereby, thus leading to a novel way to achieve liver transplantation immunotolerance.

Historical perspective of living donor liver transplantation

Living donor liver transplantation (LDLT) has gone through its formative years and established as a legitimate treatment when a deceased donor liver graft is not timely or simply not available at all. Nevertheless, LDLT is characterized by its technical complexity and ethical controversy. These are the consequences of a single organ having to serve two subjects, the donor and the recipient, instantaneously. The transplant community has a common ground on assuring donor safety while achieving predictable recipient success. With this background, a reflection of the development of LDLT may be appropriate to direct future research and patient-care efforts on this life-saving treatment alternative.

A Review of 3D Printing Technology for Medical Applications

Donor shortages for organ transplantations are a major clinical challenge worldwide. Potential risks that are inevitably encountered with traditional methods include complications, secondary injuries, and limited source donors. Three-dimensional （3D） printing technology holds the potential to solve these limitations; it can he used to rapidly manufacture personalized tissue engineering scaffolds, repair tissue defects in situ with cells, and even directly print tissue and organs. Such printed implants and organs not only perfectly match the patient＇s damaged tissue, hut can also have engineered material microstructures and cell arrangements to promote cell growth and differentiation. Thus, such implants allow the desired tissue repair to he achieved, and could eventually solve the donor-shortage problem. This review summarizes relevant studies and recent progress on four levels, introduces different types of biomedical materials, and discusses existing problems and development issues with 3D printing that are related to materials and to the construction of extracellular matrix in vitro for medical applications.

Establishment of a sensitized canine model for kidney transplantation

Objective:To establish a sensitized canine model for kidney transplantation.Methods:12 male dogs were averagely grouped as donors and recipients. A small number of donor canine lymphocytes was infused into different anatomic locations of a paired canine recipient for each time and which was repeated weekly. Specific immune sensitization was monitored by means of Complement Dependent Cytotoxicity (CDC) and Mixed Lymphocyte Culture (MLC) test. When CDC test conversed to be positive and MLC test showed a significant proliferation of reactive lymphocytes of canine recipients, the right kidneys of the paired dogs were excised and transplanted to each other concurrently. Injury of renal allograft function was scheduled determined by ECT dynamic kidney photography and pathologic investigation. Results:CDC test usually conversed to be positive and reactive lymphocytes of canine recipients were also observed to be proliferated significantly in MLC test after 3 to 4 times of canine donor lymphocyte infusions. Renal allograft function deterioration occurred 4 d post-operatively in 4 of 6 canine recipients, in contrast to none in control dogs. Pathologic changes suggested antibody-mediated rejection (delayed) or acute rejection in 3 excised renal allograft of sensitized dogs. Seven days after operation, all sensitized dogs had lost graft function, pathologic changes of which showed that the renal allografts were seriously rejected. 2 of 3 dogs in control group were also acutely rejected.Conclusion:A convenient method by means of repeated stimulation of canine lymphocyte may induce specific immune sensitization in canine recipients. Renal allografts in sensitized dogs will be earlier rejected and result in a more deteriorated graft function.

Donation after cardio-circulatory death liver transplantation

The renewed interest in donation after cardio-circulatory death (DCD) started in the 1990s following the limited success of the transplant community to expand the donation after brain-death (DBD) organ supply and following the request of potential DCD families. Since then, DCD organ procurement and transplantation activities have rapidly expanded, particularly for nonvital organs, like kidneys. In liver transplantation (LT), DCD donors are a valuable organ source that helps to decrease the mortality rate on the waiting lists and to increase the availability of organs for transplantation despite a higher risk of early graft dysfunction, more frequent vascular and ischemia-type biliary lesions, higher rates of re-listing and re-transplantation and lower graft survival, which are obviously due to theinevitable warm ischemia occurring during the declaration of death and organ retrieval process. Experimental strategies intervening in both donors and recipients at different phases of the transplantation process have focused on the attenuation of ischemia-reperfusion injury and already gained encouraging results, and some of them have found their way from pre-clinical success into clinical reality. The future of DCD-LT is promising. Concerted efforts should concentrate on the identification of suitable donors (probably Maastricht category Ⅲ DCD donors), better donor and recipient matching (high risk donors to low risk recipients), use of advanced organ preservation techniques (oxygenated hypothermic machine perfusion, normothermic machine perfusion, venous systemic oxygen persufflation), and pharmacological modulation (probably a multi-factorial biologic modulation strategy) so that DCD liver allografts could be safely utilized and attain equivalent results as DBD-LT.

Ethical tensions in solid organ transplantation: The price of success

Solid organ transplantation has rapidly developed into the therapy a choice for end-stage organ failure. The expansion of its use has resulted is a large deficiency in organ supply. To address this, the field of organ transplantation has attempted to develop new strategies that would increase the availability of organs for transplant. Some of these strategies include expansion of the donor pool by increasing the number of living donors or using deceased donor organs that may be marginal or ＂expanded＂ The intent is to bring life-saving therapy to individuals in need; however, much of this expansion has been brought forward without clear prospective guidelines. This article focuses on the current disparity between organ supply and demand, and how this has impacted the use of living donors and development of the ＂expanded donor＂ concept.

High efficiency DNA delivery into swine oocytes and embryos by electronic pulse delivery (EPD)

The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification, we applied electronic pulse delivery (EPD) technology to introduce HZKb-DC DNA construct into swine eggs. Using the developed EPD ProtocolsTM, we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos, and stable DNA transfer into the swine embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large trangenic mammals.

RAPID HLA-DRB1 GENERIC TYPING BY PCR-SSP METHOD

We report a simple and rapid HLA-DRBI generic typing method, PCRSSP, which is practical and inexpensive. We use 9 sequence-specific primers and 2 group specific Primers to define the HI-ADRB1 specificities DR1,DR2, DR3, DR4,DR5,DR6, DR7, DR8, DR9 and DR10. The HLA DR3, DR5,DR6 and DR8 can be amplified by the two primers of DR3568 and DRB1. The DR6 specificity can be identified by excluding the DR3, DR5 and DR8 when the DR3568 are positive. Any individuals can be typed with some exception: the three pairs of phenotype DR3/DR3 and DR3/DR6, DR5/DRS and DR5/DR6,DR8/DR8 and DR8/DR6 cannot be discriminated from each other. We typed 106 unrelated healthy people from Beijing locations in two weeks. We think this typing method is suitable to replace the error-prone serologic HLA-DR tests in routine clinical practice, including the Prospective typing of cadaveric organ donors.

FREE GRAFT TWO-STAGE URETHROPLASTY FOR HYPOSPADIAS REPAIR

Objective To evaluate the effectiveness of free graft transplantation two-stage urethroplasty for hypospadias repair. Methods Fifty-eight cases with different types of hypospadias including 10 subcoronal, 36 penile shaft, 9 scrotal, and 3 perineal were treated with free full-thickness skin graft or (and) buccal mucosal graft transplantation two-stage urethroplasty. Of 58 cases, 45 were new cases, 13 had history of previous failed surgeries. Operative procedure included two stages: the first stage is to correct penile curvature (chordee), prepare transplanting bed, harvest and prepare full-thickness skin graft, buccal mucosal graft, and perform graft transplantation. The second stage is to complete urethroplasty and glanuloplasty. Results After the first stage operation, 56 of 58 cases (96.6%) were successful with grafts healing well, another 2 foreskin grafts got gangrened. After the second stage operation on 56 cases, 5 cases failed with newly formed urethras opened due to infection, 8 cases had fistulas, 43 (76.8%) cases healed well. Conclusions Free graft transplantation two-stage urethroplasty for hypospadias repair is a kind of effective treatment with broad indication, comparatively high success rate, less complications and good cosmatic results, indicative of various types of hypospadias repair.

Nude mice multi-drug resistance model of orthotopic transplantation of liver neoplasm and Tc-99m MIBI SPECT on p-glycoprotein

AIM: To establish a model of drug-resistant neoplasms using a nude mice model, orthotopic transplantation of liver neoplasm and sporadic abdominal chemotherapy. METHODS: Hepatocellular carcinoma cells HepG2 were cultured and injected subdermally to form the tumor-supplying mice. The orthotopic drug-resistant tumors were formed by implanting the tumor bits under the envelope of the mice liver and induced by abdominal chemotherapy with Pharmorubicin. Physical examination, ultrasonography, spiral CT and visual inspection were used to examine tumor progression. RT-PCR and immunohistochemistry were used to detect expression of mdr1 mRNA and its encoded protein p-glycoprotein (p-gp). Tc-99m sestamibi scintigraphy was performed by obtaining planar abdominal images at 20 min after injection, and the liver/heart ratios were calculated. RESULTS: Post-implantation mortality was 0% (0/25), tumor implantation success was 90% (22/25), and the rate of implanting successfully for the second time was 100% (3/3). Tumor induction using Pharmorubicin was 80% (16/20). The mdrl mRNA expression of the induced group was 23 times higher than that of the control group, and p-gp protein expression was 13-fold higher compared to the control group. The liver/heart ratio (as assessed in vivo, using Tc-99m radiography) was decreased significantly in the induced group as compared to the control group. CONCLUSION: We have established an in vivo model of mdrl in nude mice by orthotopic transplantation of liver neoplasm coupled to chemotherapy. We propose that identification of drug resistance as characterized by decreased 99mTc-ppm radiography due to enhanced clearance by p-gp may be useful in detecting in vivo drug resistance, as well as a useful tool in designing more effective therapies.