改性聚β-羟基丁酸酯大鼠同种异体肾上腺细胞移植的研究

目的　探讨以聚 β 羟基丁酸酯 (PHB)为细胞载体进行肾上腺细胞移植治疗肾上腺皮质功能不全的可行性。方法　利用PHB作为细胞载体进行肾上腺细胞移植 ,体外观察PHB对培养的大鼠肾上腺细胞增殖、激素分泌功能的影响。将种植了大鼠肾上腺细胞的PHB植入到双侧肾上腺切除的模型大鼠体内 ,观察细胞移植受鼠体内激素水平的动态变化及局部组织学变化。结果　PHB及改性材料明胶对体外培养的肾上腺细胞增殖活性 (1.87± 0 .0 3比 1.91± 0 .0 4,P >0 .0 5 )以及分泌功能均无影响 (3 6.43± 3 .65 )ng/L比(3 1.72± 2 .49)ng/L ,P >0 .0 5 )。术后 8周实验组大鼠体内皮质酮与醛固酮水平比单纯双侧肾上腺切除组的激素水平要高 (P <0 .0 5 )。材料在植入体内 1周后可见较多炎性细胞浸润 ,有大量纤维结蒂组织包绕 ;随着时间的延长 ,炎性细胞逐步减少 ,纤维结缔组织逐步消散 ,材料逐步降解 ,血管长入。结论　改性PHB大鼠同种异体肾上腺细胞移植治疗肾上腺皮质功能不全具有可行性     

聚β-羟基丁酸酯在肾上腺细胞移植中的应用

目的探讨聚β羟基丁酸酯(PHB)对肾上腺细胞增殖和分泌功能的影响及其作为细胞载体进行肾上腺细胞移植的效果。方法将肾上腺细胞接种于PHB进行培养,观察PHB对肾上腺细胞增殖、分泌功能的影响;并将接种了肾上腺细胞的改性PHB移植至大鼠大腿肌间隙,切除受者的自体肾上腺,术后观察受鼠体内激素水平的变化,并进行局部组织学观察。结果肾上腺细胞可以在PHB上生长传代,PHB对肾上腺细胞的增殖及分泌功能的影响不显著。肾上腺细胞移植后早期,受鼠的血皮质酮和醛固酮均明显下降(P<0.05),至术后8周时血皮质酮和醛固酮均明显升高,但仍低于术前正常值,与单纯切除肾上腺的对照组比较,差异有统计学意义(P<0.05);随着术后时间的延长,移植物周围浸润的炎症细胞逐渐减少,包绕移植物的纤维结缔组织逐渐消散,PHB逐步降解,血管长入。结论PHB具有良好的生物相容性,将其作为载体行肾上腺细胞移植是可行的。     

新型聚β-羟基丁酯作为血管组织工程支架材料的实验研究

目的：探讨经改性处理后的可降解生物材料聚β-羟基丁酯(PHB)作为血管组织工程支架材料与血管平滑肌细胞的细胞相容性。方法：采用体外培养的免血管平滑肌细胞接种在经胶原包埋处理后的管型PHB支架材料上，用相差显微镜和扫描电镜观察细胞的粘附和生长情况，并行HE染色观察。结果：免血管平滑肌细胞在改性后的管型PHB支架材料上粘附生长良好，扫描电镜下可见细胞生长融合成片状，HE染色示细胞在PHB材料上生长良好。结论：改性后的聚β-羟基丁酯材料与兔血管平滑肌细胞有较好的生物相容性。     

兔血管平滑肌细胞和聚羟基丁酯(PHB)的细胞相容性实验研究

目的：探索血管平滑肌细胞和新型可降解材料聚羟基丁酯（PHB）的细胞相容性，为组织工程血管的构建寻找理想的支架材料。方法：将组织块法体上培养与兔血管平滑肌细胞种植在PHB膜片和PHB三维微孔支架上，在相差显微镜下观察细胞的粘附和生长情况。用MTT法测定细胞粘附率和细胞增殖指数，复合培养7天后进行扫描电镜观察并用流式细胞仪（FCM）的测定细胞周期，DNA指数。结果：兔血管平滑肌细胞在PHB膜片上粘附率为77％，细胞增殖符合细胞的生长曲线，在PHB三维微孔支架上生长情况良好，并被证实为二倍体细胞。结论：兔血管平滑肌细胞和聚羟基丁酯（PHB）的细胞相容性较好，但细胞与材料间的粘附有待进一步改善。     

聚羟基丁酸酯可吸收缝线生物相容性的实验室研究

目的：研究聚羟基丁酸酯（Polyhydroxybutyrate,PHB）可吸收缝线的生物相容性。方法：①将成纤维细胞进行体外培养,传代后与PHB可吸收缝线复合培养,设与经聚氰基丙烯酸酯处理过的PHB可吸收缝线复合培养为对照组,观察细胞生长情况;②行扫描电镜检查观察细胞在丝线上的生长情况;③以MTT法检测复合培养后的细胞活性。结果：①实验组细胞生长良好,对照组细胞漂浮,收缩成团;②扫描电镜下见细胞在丝线上贴附生长良好;③MTT法检测实验组对细胞增殖无明显影响。结论：PHB可吸收缝线具有良好的生物相容性。     

以聚β-羟基丁酸酯为支架的肾上腺细胞移植形态学研究

目的探讨以聚β-羟基丁酸酯(PHB)为支架的肾上腺细胞移植后的形态学变化。方法分离Wistar大鼠的肾上腺细胞,接种至改性的PHB上,然后移植至切除双侧肾上腺的大鼠的后肢肌间隙中,并设单纯双侧肾上腺切除的对照组及假手术组。观察术后第2、4、8周时移植局部的组织学变化,免疫组化染色观察移植局部CD3表达。结果组织学观察显示,随着时间的推移,移植物的组织反应逐渐变小,PHB逐渐降解,肾上腺细胞趋向组织化;超微结构观察显示,移植的肾上腺细胞保持着同正常肾上腺细胞相似的结构;免疫组织染色显示,移植的肾上腺细胞组织化后其CD3的表达与正常肾上腺组织相似(P>0.05)。结论以PHB为支架的肾上腺细胞移植,移植物在体内可存活,且呈现明显的组织化,移植的肾上腺细胞与正常肾上腺细胞的超微结构基本一致。     

尿道种子细胞与生物可降解材料的生物相容性研究

目的 观察生物可降解材料聚β-羟基丁酸酯(PHB)与种子细胞即兔膀胱移行上皮细胞及平滑肌细胞间的生物相容性,探讨其作为种子细胞载体构建组织工程化尿道的可行性.方法 经过传代培养,分别在PHB与上皮细胞共同培养2、7 d,与平滑肌细胞共同培养1、5 d,每组各取6个样本,用噻唑蓝(MTT)比色法检测细胞的增殖情况,并利用扫描电镜检测细胞在材料上的空间生长情况,评估移行上皮细胞和平滑肌细胞与聚β-羟基丁酸酯的生物相容性.结果 平滑肌细胞能够在该材料上正常生长,各组间A值差异无统计学意义(P>0.05);而移行上皮细胞在该材料上只有少量生长,且实验组与对照组间A值差异有统计学意义(P<0.05),而A、B两实验组间A值差异无统计学意义(P>0.05).结论 聚β-羟基丁酸酯与平滑肌细胞具有良好的生物相容性,但其对移行上皮细胞的生长具有一定的抑制作用,生物相容性不甚理想.     

以聚β-羟基丁酸酯为载体的肾上腺细胞移植实验研究

目的 探讨以聚β-羟基丁酸酯(PHB)作为细胞载体进行肾上腺细胞移植的效果。方法Wistar大鼠切除双侧肾上腺后，分别行单纯肾上腺细胞移植和以PHB为载体的肾上腺细胞移植，术后动态观察受者的血清皮质酮和醛固酮水平以及移植物组织学形态。结果 肾上腺细胞移植后，大鼠血清皮质酮和醛固酮的水平升高，以PHB为载体的细胞移植组升高更加显著，形态学证实PHB载体组的肾上腺细胞生长良好。结论 以PHB为载体的肾上腺细胞移植比单纯肾上腺细胞移植效果更好。     

骨组织工程快速成型支架改性的相关研究

目的探讨胶原杂化及磷灰石表面沉积改性后的聚乳酸一羟基乙酸共聚物／β-磷酸三钙（PLGA／β—TCP）作为快速成型支架应用于骨组织工程的可行性。方法使用骨髓基质干细胞对胶原杂化及磷灰石表面沉积改性后的PLGA／β-TCP的生物相容性进行评估，通过扫描电镜观察改性后材料的表面特性及细胞与材料复合的形态学特征；细胞与材料复合后的繁殖与分化能力分别使用细胞计数及碱性磷酸酶定量方法进行评估。结果通过对亲水性、细胞增殖能力及碱性磷酸酶测定证实改性后的PLGA／β-TCP快速成型支架较单纯材料其亲水性及生物相容性有明显提高（P〈0．05）。结论胶原杂化及磷灰石表面沉积改性后的PLGA／β—TCP快速成型支架可作为三维支架应用于骨组织工程。     

生物可降解材料聚β-羟基丁酯对兔骨髓基质细胞生物学特性的影响

目的：检测生物可降解材料聚β-羟基丁酯(PHB)对兔骨髓基质细胞(BMSC)形态、生长、附着及增殖的影响。方法：将家兔BMSC进行体外培养，以诱导剂(β-甘油磷酸钠，地塞米松，L-抗坏血酸)使其向成骨细胞分化，行碱性磷酸酶染色并计算阳性率，将PHB与成骨细胞样细胞进行体外复合培养，用倒置显微镜及扫描电镜观察PHB对BMSC的形态、生长、附着及增殖的影响。结果：在诱导剂的作用下BMSC向成骨细胞分化，碱性磷酸酶染色呈阳性，其阳性率随培养时间延长而增加。PHB与成骨细胞样细胞进行体外复合培养后显示PHB对BMSC的形态、生长、附着及增殖均无不良影响。结论：PHB具有良好的生物相容性，可望与骨髓复合移植修复骨缺损。     

Intradermal cell transplantation in soluble collagen

Intradermal, as opposed to subcutaneous, cell transplantation was previously shown to be advantageous for tumor cell growth, but this site has not been used for transplantation of normal nonneoplastic cells. In preliminary experiments we found that it was difficult to control the size and shape of transplants when we injected dissociated cells intradermally. This problem was solved by placing cells in nongelled, pepsin-solubilized collagen prior to injection. This technique permitted the successful transplantation of normal bovine adrenocortical cells and of neoplastic cells (3T3 cells secreting FGF) in scid mice. Primary bovine adrenocortical cells formed functional vascularized tissue and the transplants rescued the animals from the lethal effects of adrenalectomy. The histological structure of transplant tissues resembled that previously observed when cells were transplanted in the subrenal capsule space. We also used a line of 3T3 cells that has been genetically modified to secrete a form of acidic FGF. When transplanted intradermally in collagen, they formed rapidly enlarging masses of cells that could easily be palpated beneath the skin of the animal. Intradermal injection of cells in pepsin-solubilized collagen is a simple and reliable technique for transplanting normal primary cells and preneoplastic cells. The ability to grow both types of cells in an easily accessible site allows less invasive monitoring of growth, angiogenesis, and other features of the transplant.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

目的 探讨大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植治疗肾上腺功能不全的可行性.方法 分离SD大鼠(出生3 d内的乳鼠)的肾上腺细胞,置于自制鼠尾Ⅰ型胶原凝胶中培养,1周后移植到双侧肾上腺切除的成年雄性SD大鼠肾被膜下.术后每周取血1次,测量血浆皮质酮和醛固酮的水平;移植8周后处死大鼠,观察移植物的组织学特点;肾上腺细胞的增殖情况及CYP11B1和CYP11B2蛋白的表达情况;并与单纯肾上腺细胞移植组和正常对照组相比较.结果 大鼠移植经胶原凝胶三维培养的肾上腺细胞后,80%(12/15)存活达8周;手术后血浆皮质酮水平显著高于同期单纯肾上腺细胞移植组,移植6～8周时,与正常对照组已无显著性差异;其醛固酮水平术后恢复缓慢,与单纯肾上腺细胞移植组的差异无统计学意义;移植后肾上腺细胞在胶原内生长良好,增殖率高,95%为束状带细胞,表达CYP11B1,而表达CYP11B2的球状带细胞仅占5%,未见炎症细胞浸润.结论 肾上腺细胞经胶原凝胶三维培养后移植能形成有功能的血管化肾上腺组织,长期效果理想,有望成为临床治疗肾上腺功能不全的有效方法.     

A murine model of allogeneic adrenocortical cell transplantation: perspectives for the treatment of Addison's disease in humans

BACKGROUND: Hormone substitution for the treatment of adrenocortical insufficiency (Addison's disease) does not adequately substitute the hormone peaks required in stress situations. Therefore, allogeneic transplantation of adrenal cortex could offer an intriguing alternative. METHODS: Major histocompatibility complex (MHC) class I transgenic mice were used for the implementation of an animal model of adrenocortical cell transplantation in adrenalectomized mice. K(b)-transgenic cells and allogeneic adrenocortical cells were cocultured in mixed lymphocyte cultures to examine the alloimmune response. Lymphocytes from T-cell receptor transgenic mice and normal allogeneic mice served as responder cells. The effect of corticosteroids secreted by adrenocortical cells was antagonized by the steroid receptor antagonist mifepristone (RU486). RESULTS: In vitro coculture experiments showed that MHC class I disparate adrenocortical cells failed to activate B10.BR and T-cell receptor transgenic lymph node cells. In the presence of mifepristone this inhibitory effect was antagonized, resulting in strong lymphocyte proliferation. Activation of B10.BR lymphocytes by K(b)-disparate spleen cells was also abolished in the presence of adrenocortical cells. This effect, however, could not be reversed by mifepristone. CONCLUSIONS: In vitro, the presence of adrenocortical cells potently suppressed allogeneic immune responses. This effect was only in part due to the secretion of corticosteroids, pointing to an additional immunomodulatory property of adrenocortical cells.     

Suramin and the human adrenocortex: results of experimental and clinical studies.

Suramin, a sulfonated drug, has been used successfully in the treatment of inoperable adrenocortical cancer. This study was undertaken to investigate the effects of suramin on the basal and the adrenocorticotropin-stimulated cortisol and pregnenolone secretion and on the proliferation of primary monolayer cultures of normal human adrenocortical cells. Suramin decreases basal and adrenocorticotropin-stimulated cortisol secretion in a dose-dependent manner (p less than 0.05 from 0.3 mmol/L upward). At a suramin concentration of 3 mmol/L cortisol, secretion was inhibited by 70% +/- 4% in adrenocorticotropin-stimulated cells and by 42% +/- 6% in unstimulated cells. The proliferation of adrenocortical cells in response to fetal calf serum was inhibited by suramin at concentrations from 0.3 mmol/L upward, maximal suppression (71% +/- 6%; p less than 0.05) being observed at a concentration of 10 mmol/L. Neither down-regulation of cortisol secretion nor inhibition of adrenocortical cell proliferation was caused by toxicity of the compound, as could be shown by adrenocorticotropin-restimulating cortisol secretion in suramin-treated cells. The results indicate that suramin exerts an inhibitory influence on the cortisol secretion and the proliferation of normal human adrenocortical cells and may be useful in treating adrenocortical cancer.     

晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响

目的：探讨晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响,以期揭示晚期糖化终末产物在骨质疏松发病中的作用。方法：用体外制备的AGE-牛血清白蛋白（AGE-BSA）加入人成骨细胞培养体系,观察不同浓度AGE和不同作用时间,对人成骨细胞增殖及分泌白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的影响。结果：加入AGE-BSA24h后,低浓度抑制成骨细胞增殖;培养48、72h后,浓度依赖性的促增殖作用增加。加入AGE-BSA48h,100μg/ml AGE-BSA促进成骨细胞分泌IL-6。200μg/ml-100μg/ml对IL-6及TNF-α的分泌均无显影响。结论：晚期糖化终末产物对人成骨细胞表现出在较高浓度时,仍具有很强的促增殖活性,提示人对高浓度AGE的耐受性增强。AGE在较高浓度对人成骨细胞分泌IL-6和TNF-α无显影响。提示由随龄增加的AGE在老年人引起的成骨细胞介导的骨吸收没有明显增加,与老年性骨质疏松为低转换型结果相一致。     

胰岛素类似物生长因子1受体基因沉默对人肾上腺皮质癌细胞株SW13增殖凋亡的影响

目的探讨胰岛素类似物生长因子1受体(IGF1R)基因沉默对人类肾上腺皮质癌细胞株SW13增殖及凋亡的影响。方法依据人类IGF1R的cDNA序列合成其特异性SiRNA,通过脂质体将其转入SW13细胞,24h后通过实时定量Real time PCR检测SiRNA对靶基因mRNA下调作用,同时使用Western blot检测其靶蛋白的表达。通过MTT法测定转染后细胞增殖活性,流式细胞仪测定细胞凋亡状态。结果3种不同的针对IGF1R的SiRNA能够有效下调SW13细胞IGF1R mRNA,而通过Western blot也发现在蛋白水平出现类似结果。IGF1R基因沉默能够有效抑制SW13细胞增殖以及诱导细胞凋亡。结论IGF1R基因沉默能够诱导肾上腺皮质癌细胞凋亡,抑制其增殖,有望为肿瘤靶向治疗在ACC中应用提供相应的理论依据。     

Enhanced spontaneous antibody response after coronary artery bypass surgery

Purpose

Cells spontaneously secreting immunoglobulins can be seen in the Wood one week after open-heart surgery. The purpose of this study was to measure the antibody specificities of activated cells.

Methods

Immune responses were studied preoperatively and on the seventh postoperative day in 18 patients undergoing elective coronary artery bypass surgery. The number of cells secreting adenovirns. measles, rubella and tetanus antigen specific antibodies spontaneously and induced by pokeweed mitogen PWM (ASCs) as well as the total number of cells secreting IgG. IgM and IgA (ISCs) were studied using an enzyme-linked immunospot (ELISPOT) assay. Spontaneous as well as phytohaemagglutinin (PUA)- and pokeweed mitogen (PWM)-induced lymphocyte proliferation was also measured.

Results

The number of cells spontaneously secreting IgG. IgM and IgA antibodies was increased on the seventh day after coronary bypass surgery, against adenovirus, measles, rubella and tetanus as well as the total number of cells secreting immunoglobulins IgG, IgM and IgA (P < 0.05/0.001). By contrast, only slight fluctuation was seen in the numbers of cells secreting antibodies after PWM stimulation. Spontaneous lymphocyte proliferation was also increased. PUA proliferative responses were depressed and PWM responses were not changed on the seventh postoperative day compared with preoperative values.

Conclusion

Coronary artery bypass surgery caused marked polyclonal B cell activation demonstrated by an increase of cells producing spontaneously antibodies against virus antigens and tetanus toxoid. This activation could not be intensified by PWM stimulation.     

Allogeneic adrenocortical transplantation: glucocorticosteroid-independent immunomodulatory properties of adrenal cortex cells

BACKGROUND: Hormone substitution for the treatment of adrenocortical insufficiency does not adequately substitute the physiologic circadian secretion of corticosteroids and leads to long-term sequelae and reduced quality of life. The lack of adaptation to physical and psychologic stress situations may lead to life-threatening Addison's crises. Allogeneic transplantation of adrenal cortex could offer an intriguing alternative. Adrenocortical grafts were demonstrated to proliferate and produce corticosteroids in physiologic concentrations after transplantation. METHODS: K -transgenic murine lymphocytes and allogeneic adrenal cortex cells were cocultured in mixed lymphocyte reactions to examine the alloimmune response; lymphocytes from T-cell receptor transgenic mice and normal mice, respectively, served as responder cells. The effects of corticosteroids secreted by adrenocortical cells were antagonized by the steroid receptor antagonist mifepristone, whereas the impact of cell-cell interactions was differentiated with transwell culture systems. RESULTS: Coculture of adrenal cortex cells in mixed lymphocyte reactions markedly suppressed lymphocyte proliferation. Transwell cultures demonstrated that adrenocortical cells exerted their effects by a soluble factor that was only partially antagonized by mifepristone. CONCLUSION: In vitro, the presence of adrenocortical cells potently suppressed allogeneic immune responses. This effect was not exclusively the result of the secretion of corticosteroids, indicating an additional immunomodulatory property of adrenocortical cells.