微囊化人头皮毛乳头细胞体外培养及异种移植

目的探讨微囊化人头皮毛乳头细胞体外培养及异种移植的可行性,并对海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate,APA）微囊与海藻酸钠-BaCi2（barium-alginate,BA）微囊的物理、生物性能进行评价.方法采用“一步酶消化法”分离、培养人头皮毛乳头细胞,分别用APA微囊与BA微囊包裹.对两种微囊的生物相容性、机械强度、免疫隔离效果及微囊内细胞活性进行比较.结果APA微囊生物相容性优于BA微囊（P＜0.01）,但机械强度低于BA微囊（P＜0.01）;成囊后短期BA微囊内细胞活性高于APA微囊（P＜0.01）,但APA微囊内细胞活性增高较快（P＜0.05）.在微囊完整、表面无纤维化时,两种微囊均可起到良好的免疫隔离作用.结论微囊化人头皮毛乳头细胞可在体外及异种体内培养.综合评价APA微囊与BA微囊的利弊,在不同情况下选择不同的成囊方式是必要的.     

微囊豚鼠肝细胞培养及其细胞免疫屏障作用的观察

目的 观察海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化肝细胞的生物学功能及对免疫细胞的隔离效应。方法 用胶原酶门腔静脉灌注法分离豚鼠肝细胞,测定微囊化肝细胞及游离肝细胞培养上清白蛋白水平,用自然杀伤细胞(NK)细胞的细胞毒实验来评价微囊的免疫隔离效应。结果 微囊包裹对肝细胞的活率无明显影响,微囊化肝细胞与裸露肝细胞白蛋白分泌水平差异无显著性(P>0.05),NK细胞对K562靶细胞的细胞毒实验表明微囊可有效地保护囊内细胞不受NK细胞的杀伤作用。结论 APA微囊化肝细胞可保持良好的生物活性,APA微囊对细胞免疫具有免疫隔离作用。     

微囊化胰岛移植的研究进展

免疫隔离膜微囊化胰岛移植，通过一个人造屏障将胰岛与宿主的免疫系统隔离开业，为解决排斥反应开辟了新的思路和途径。在糖尿病动物模型上获得了令人鼓舞的效果，本文就海藻酸钠微囊的制备、免疫屏障作用、移植数量和移植后纤维化进行了探讨。     

海藻酸钠纯度对微囊化肝细胞腹腔移植中微囊纤维化的影响

目的　研究海藻酸钠纯度对微囊纤维化的影响。　方法　采用逐步过滤和氯仿 /丁醇萃取的方法纯化海藻酸钠 ,用纯化和未纯化的海藻酸钠制作两种微囊化肝细胞分别给两组大鼠 (各2 0只 )腹腔移植 ,观察不同时期微囊的回收率、微囊纤维化、囊内肝细胞的功能和结构。用3 H TdR掺入法测定纯度对淋巴细胞转化率的影响。　结果　纯化组的微囊回收率较未纯化组明显增高 (P <0 0 1)。纯化组移植 1个月后大部分肝细胞形态及酶组化染色正常。未纯化组移植后 1个月内肝细胞破坏。移植 6个月时 ,纯化组微囊仍保持完整光滑 ;而未纯化组移植 1个月后微囊全部纤维化。纯化的海藻酸钠对人类淋巴细胞转化率明显低于未纯化组。　结论　纯化的海藻酸钠能明显降低微囊的纤维化程度     

海藻酸钠-氯化钡微囊对大鼠胰岛体外胰岛素分泌功能的影响

目的　观察海藻酸钠 -氯化钡微囊对大鼠胰岛体外胰岛素分泌功能有无影响。方法　以海藻酸钠和氯化钡为材料 ,采用气体吹喷制囊法将新鲜分离纯化的大鼠胰岛制成微囊化胰岛 ,取空微囊、微囊化大鼠胰岛与未微囊化大鼠胰岛各 5 0 0只 ,分为 10份 ,置于培养板中培养 ,用放免法测定并比较第 2、4、6 d培养液中基础胰岛素浓度。结果　空微囊组第 2、4、6 d的基础胰岛素平均浓度均为 0 nm ol/ 1/ 5 0 ,微囊化大鼠胰岛组第 2、4、6 d的基础胰岛素平均浓度为 5 .179、5 .80 6、5 .5 5 8nm ol/ 1/5 0只 ,未微囊化大鼠胰岛组第 2、4、6 d的基础胰岛素平均浓度为 5 .4 4 1、6 .0 80、5 .4 6 8nmol/ 1/ 5 0只 ,后两者差异无显著性意义 (P>0 .0 5 )。结论　海藻酸钠 -氯化钡微囊对大鼠胰岛体外胰岛素分泌功能无影响     

壳聚糖相对分子质量对微囊及包裹肝细胞活性的影响

目的 观察壳聚糖相对分子质量对微囊机械强度和通透性及包裹肝细胞活性的影响.方法 通过微囊搅拌实验比较中、低分子量壳聚糖形成的微囊的机械强度,比较2种微囊对异硫氰酸荧光素标记的牛血清白蛋白(FITC-BSA)通透性,及细胞染色实验判断2种微囊包裹小鼠原代肝细胞活性的区别.结果 微囊搅拌100 min后中分子量壳聚糖微囊破损率100%,低分子量壳聚糖微囊破损率5%,两种微囊机械强度差异有统计学意义(P＜0.05),微囊溶液中加入HTC-BSA15 min后,低分子量壳聚糖微囊内荧光强度为4 AU,中分子量壳聚糖微囊内荧光强度为1.5 AU,两种微囊对FITC-BSA的通透性差异有统计学意义(P＜0.05),低分子量壳聚糖形成的微囊包裹肝细胞培养1周后细胞染色实验显示微囊中活肝细胞数为100%,中分子量壳聚糖形成的微囊包裹的活肝细胞数约为40%,两者差异有统计学意义(P＜0.05).结论 低分子量壳聚糖相对于中分子量壳聚糖更适合于作微囊的材料.

Abstract：

Objective To study the influence of molecular weight of chitosan on microcapsules and hepatocytes in microcapsules. Methods The mechanical strength, permeability to fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA) and activity of hepatocytes in microcapsules were compared between two kinds of microcapsules made by low and middle molecular weight chitosan. Results After 100 min of stirring microcapsules, all middle molecular weight chitosan microcapsules were damaged, 5% low molecular weight chitosan microcapsules were damaged. There was significant difference in breakage rate of the mechanical strength between two microcapsules (P ＜ 0. 05). Fifteen min after addition of FITCBSA into microcapsule solution, fluorescence intensity in the low molecular weight chitosan microcapsules was 4 AU, and that in middle molecular weight of chitosan microcapsules was 1. 5 AU, suggesting there was significant difference in permeability to FITC-BSA between two kinds of microcapsules (P ＜ 0. 05).One week after culture of microencapsulated hepatocytes, staining test showed that 100% of liver cells in low molecular weight chitosan microcapsules were alive, while the number was about 40% in middle molecular weight chitosan microcapsules (P ＜ 0. 05). Conclusion Low molecular weight chitosan is more suitable as materials of microcapsules than molecular weight chitosan.     

壳聚糖相对分子质量对微囊及包裹肝细胞活性的影响

Objective To study the influence of molecular weight of chitosan on microcapsules and hepatocytes in microcapsules. Methods The mechanical strength, permeability to fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA) and activity of hepatocytes in microcapsules were compared between two kinds of microcapsules made by low and middle molecular weight chitosan. Results After 100 min of stirring microcapsules, all middle molecular weight chitosan microcapsules were damaged, 5% low molecular weight chitosan microcapsules were damaged. There was significant difference in breakage rate of the mechanical strength between two microcapsules (P ＜ 0. 05). Fifteen min after addition of FITCBSA into microcapsule solution, fluorescence intensity in the low molecular weight chitosan microcapsules was 4 AU, and that in middle molecular weight of chitosan microcapsules was 1. 5 AU, suggesting there was significant difference in permeability to FITC-BSA between two kinds of microcapsules (P ＜ 0. 05).One week after culture of microencapsulated hepatocytes, staining test showed that 100% of liver cells in low molecular weight chitosan microcapsules were alive, while the number was about 40% in middle molecular weight chitosan microcapsules (P ＜ 0. 05). Conclusion Low molecular weight chitosan is more suitable as materials of microcapsules than molecular weight chitosan.     

壳聚糖相对分子质量对微囊及包裹肝细胞活性的影响

Objective To study the influence of molecular weight of chitosan on microcapsules and hepatocytes in microcapsules. Methods The mechanical strength, permeability to fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA) and activity of hepatocytes in microcapsules were compared between two kinds of microcapsules made by low and middle molecular weight chitosan. Results After 100 min of stirring microcapsules, all middle molecular weight chitosan microcapsules were damaged, 5% low molecular weight chitosan microcapsules were damaged. There was significant difference in breakage rate of the mechanical strength between two microcapsules (P ＜ 0. 05). Fifteen min after addition of FITCBSA into microcapsule solution, fluorescence intensity in the low molecular weight chitosan microcapsules was 4 AU, and that in middle molecular weight of chitosan microcapsules was 1. 5 AU, suggesting there was significant difference in permeability to FITC-BSA between two kinds of microcapsules (P ＜ 0. 05).One week after culture of microencapsulated hepatocytes, staining test showed that 100% of liver cells in low molecular weight chitosan microcapsules were alive, while the number was about 40% in middle molecular weight chitosan microcapsules (P ＜ 0. 05). Conclusion Low molecular weight chitosan is more suitable as materials of microcapsules than molecular weight chitosan.     

细胞微囊技术在再生医学领域的应用

细胞微囊具有良好的免疫隔离作用.微囊体内移植仍面临炎性细胞浸润和纤维化包襄的难题,微囊制备、材料选择及体内移植部位对提高其生物相容性尤为重要.目前细胞微囊化技术在再生医学领域的应用受到广泛重视,转基因干细胞微囊化后可持续分泌组织再生所需生长因子,微囊创造的微环境也为干细胞分化提供了有利场所.     

微囊化胰岛移植的研究进展(文献综述)

免疫隔离膜微囊化胰岛移植,通过一个人造屏障将胰岛与宿主的免疫系统隔离开来,为解决排斥反应开辟了新的思路和途径.在糖尿病动物模型上获得了令人鼓舞的效果,本文就海藻酸钠微囊的制备、免疫屏障作用、移植数量和移植后纤维化进行了探讨.     

Immunoisolated chromaffin cells implanted into the subarachnoid space of rats reduce cold allodynia in a model of neuropathic pain: a novel application of microencapsulation technology

Intrathecal transplants of adrenal medullary chromaffin cells relieve chronic pain by secreting catecholamines, opioids, and other neuroactive substances. Recently, macrocapsules with semipermeable membranes were used to isolate immunologically xenogenic chromaffin cells, but the poor viability in vivo of the encapsulated chromaffin cells limited the usefulness of this method. In this study, we used a novel method of encapsulation to increase the viability of chromaffin cells. We found that microencapsulated chromaffin cells that were implanted into the subarachnoid space of rats relieved cold allodynia in a model of neuropathic pain. Furthermore, microencapsulated chromaffin cells were morphologically normal and retained their functionality. These findings suggest that the intrathecal placement of microencapsulated chromaffin cells might be a useful method for treating chronic pain.     

循环肿瘤细胞检测技术及其临床应用研究进展

肿瘤细胞脱落、侵袭并进入血液循环是实现肿瘤转移的最初阶段,并为最终形成临床转移灶提供可能.深入研究循环肿瘤细胞有助于对肿瘤转移机制的了解,可为抗转移治疗提供依据.本文将对循环肿瘤细胞的检测技术及临床意义作一综述.     

微囊化人头皮毛乳头细胞诱导小鼠耳毛囊再生的研究

目的 观察人头皮毛乳头细胞海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate，APA）微囊是否具备诱导小鼠耳部毛囊再生的功能；寻找理想的微囊直径。方法 以APA微囊包裹体外分离培养的人头皮毛乳头细胞；将毛乳头细胞微囊移植至小鼠耳部皮下，6周后局部取材行组织学检查；共聚焦显微镜下观察葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式，并对比相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度，综合分析确定最佳的微囊直径。结果 组织学检查显示：移植部位皮下有密集的同心圆状毛囊结构形成，其数量、大小、分化程度等与对照组明显不同。荧光素以同心圆状、逐层渗透的方式向APA微囊中扩散；相同时间内荧光强度比较：小囊组〉中囊组〉大囊组。结论 微囊化毛乳头细胞具备诱导毛囊再生的生理功能；微囊理想的直径是400μm。     

Effect of high energy shock waves on tumor cells

Summary Exposure of bladder tumor cell strain HT-1197, chronic bonemarrow leukemic cell strain K-562, and African green-turtle normal kidney cell strain Vero to high energy shock waves resulted in ultrastructural changes and a reduction in cell viability as determined by ³H-thymidine incorporation assay and flowcytometer. K-562 was the most sensitive while Vero was the most resistant to the high energy shock wave. By flowcytometry using anti BrdU antibody, described K-562 in the S phase was found to be inhibited by the exposure. Electron microscopy revealed destruction of microvilli over the cell surface and swollen mitochondria in K-562 and HT-1197. These effects were related to the number of high energy shock wave exposures. Our study demonstrates that a high energy shock wave has an anti-tumor effect in vitro.     

Differential sensitivity of hormone-responsive and unresponsive human prostate cancer cells (LNCaP) to tumor necrosis factor

Summary Two sublines, the hormone-sensitive LNCaP-FGC and the insensitive LNCaP-r (resistant) carcinoma cell lines, originating from the parental human prostatic carcinoma cell line LNCaP were tested for sensitivity to human tumor necrosis factor- (TNF) using the MTT assay. Irrespective of the culture conditions, i.e., whether FGC cell growth was hormone stimulated or hormone deprived, a clear dose-related response was observed between the concentration of TNF (range: 5–5000 U/ml) in the culture medium and the percentage of growth inhibition. In medium containing androgen-depleted serum, in which FGC cells showed reduced proliferative activity, the percentage of inhibition by a concentration of 100 U/ml TNF was substantially higher than that found in hormone-stimulated cells (90% and 60%, respectively). In contrast to the FGC cells, the hormone-insensitive LNCaP-r cells were almost completely resistant to the action of TNF. Growth of the FGC cells was almost completcly inhibited, whereas growth of the LNCaP-r cells was retarded with only 20% at dosages up to 5000 U/ml. This substantial difference in TNF responsiveness could not be ascribed to differences in TNF-binding capacity, as both the FGC and LNCaP-r cells were found to contain identical numbers of TNF-receptors (approximately 1000 sites/cell). A possible association between hormone responsiveness and TNF sensitivity is suggested for these LNCaP sublines.This study was supported in part by the Stichting Urologisch Wetenschappelijk Onderzoek (SUWO)This paper was selected for publication in Urological Research from the program of the 1991 meeting of the European Society of Urological Oncology and Endocrinology (ESUOE)     

The inhibition of tumor cell adhesion on human mesothelial cells (HOMC) by phospholipids in vitro

Background and aims Intraperitoneal tumor cell adhesion to extracellular matrix and to mesothelial cells mediated by integrins is an important step in developing peritoneal carcinosis. In former animal studies, we could demonstrate that intraperitoneal treatment with a new phospholipid (PL) emulsion significantly reduces the amount of peritoneal carcinosis by adhesion prevention. This in vitro study tries to elucidate the influence of phospholipids on cells of the human gastric cancer cell line (NUGC-4) and the human rectal cancer cell line (HRT-18) adhering to mesothelial cells (HOMC) in a monolayer culture in vitro. Materials and methods HOMC cells were derived from omentum majus from patients undergoing elective abdominal surgery. Three passages of both cancer cell lines (NUGC-4 and HRT-18) were used. 1×10⁵/100 μl (HRT-18) or 1.2×10⁵/100 μl (NUGC-4) cells, according to forgoing dilution series, were pretreated with different concentrations of phospholipid emulsion (0.05, 0.1, 0.5, 0.75, 1% PL) stained with cell tracker chloromethyl-benzamidodialkylcarbocyanine (CM-DIL) and seeded into each well on the mesothelial monolayer. After 90 min, the number of adherent cells was counted by fluorescence microscopy at 530 and 620 nm. Additionally, flow cytometric analysis of integrin α3 and β1 expression on the tumor cell surface after treatment with phospholipids was completed. Results We found a dose dependent effect of phospholipids on both tumor cell lines causing a reduction of cell–cell adhesion. Already low concentrations of phospholipids (PL 0.5) had a significant influence. The mean cell count could be reduced from 234±12/mm² in controls to 124±41/mm² (PL 0.5; NUG-4) and from 295±49/mm² to 169±29/mm² (PL 0.5; HRT-18), respectively. Additionally, the integrin α3 and β1 expression on both cell lines could be reduced. Conclusion Our results within the scope of published data indicate that adhesion prevention is capable to reduce peritoneal carcinosis. Best of Forum Papers presented at the Annual Meeting of the German Society of Surgery, 2–5 May 2006, Berlin, Germany     

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.B. Frankenberger and S. Regn made equal contributions to these studies     

热休克蛋白-多肽复合物/树突状细胞疫苗的研究

目的　探讨从肺癌组织中提取的热休克蛋白GP96 多肽复合物 (GP96)负载树突状细胞 (DC)疫苗在小鼠体内的抗肿瘤作用。方法　自小鼠肺腺癌LA795肿瘤组织中提取GP96,自615小鼠外周血中提取单个核细胞体外培养DC ,共孵育制备疫苗 ,分别以GP96(5 μg/只 )、DC(5×10 5个 /只 )和GP96/DC(DC5× 10 5个 /只 ,培养时加入 5 μgGP96)免疫小鼠后接种肿瘤 ,与未免疫组比较成瘤率、肿瘤体积及生存时间。同时以联合免疫缺陷小鼠 (SCID )进行类似实验 ,肿瘤组织为人肺腺癌PAa ,DC来源于人外周血 ,分组剂量同上。结果　 615组小鼠成瘤率分别为 10 0 %、10 0 %、10 0 %和 75 % ,肿瘤体积为 (6.46± 1.47)cm3 ,(0 .70± 0 .62 )cm3 ,(0 .64± 0 .49)cm3 和(0 .3 2± 0 .3 0 )cm3 ,生存时间为 5 2、5 1.5、5 8.5和 77d ,SCID组小鼠成瘤率分别为 10 0 %、10 0 %、10 0 %和 87.5 % ,肿瘤体积为 (0 .75± 0 .3 0 )cm3 ,(0 .17± 0 .0 6)cm3 ,(0 .15± 0 .0 8)cm3 和 (0 .0 8±0 .0 3 )cm3 ,生存时间为 3 7、5 2、5 2 .5和 77d。结论　GP96/DC疫苗有更明显的抑制肿瘤生长的作用。     

树突状细胞与胃癌细胞融合的肿瘤疫苗研究

目的以肿瘤细胞与树突状细胞(dendriticcells,DCs)相融合使融合细胞能将肿瘤抗原递呈给T细胞,并提供T细胞激活所必需的第二信号,借此激活机体的抗肿瘤免疫。方法通过PEG法将鼠源性胃癌细胞MFC与小鼠DC进行融合(T/DC),融合后经50Gy电子线照射,以1×106肿瘤疫苗于小鼠皮下注射免疫2次,间隔1周后接种,后一次免疫后1周于小鼠皮下接种5×105MFC胃癌细胞。肿瘤细胞接种3周后处死实验动物,测量肿瘤大小、外周血NK活性和脾细胞CTL活性。结果肿瘤接种后一周对照组小鼠皮下均有肿瘤生长,而T/DC免疫组均无皮下可触及的肿瘤(10/10),10天后T/DC免疫组才有4/10只鼠皮下出现可触及的肿瘤,3周后对照组肿瘤体积明显大于T/DC免疫组。经T/DC免疫后小鼠生存期明显较对照组延长,对照组于肿瘤接种后2周开始有小鼠衰竭死亡,至肿瘤接种后3周对照组仅有3只存活,T/DC免疫组有8只鼠存活,并仍有3只无肿瘤生长。经T/DC免疫后,小鼠NK活性和肿瘤特异性CTL活性均明显提高,T/DC免疫组和对照组MFC特异性CTL活性分别为30.09%和7.12%。结论肿瘤细胞鄄DC融合的肿瘤疫苗可通过加强抗原递呈激活肿瘤特异性CTL而具明显的抗肿瘤活性。     

肿瘤蛋白D52在肿瘤发生发展中作用及其在肿瘤诊疗中潜在应用价值

肿瘤蛋白D52(TPD52)在乳腺癌、前列腺癌、肺癌、卵巢癌和血液系统恶性病变等多种肿瘤中高表达,可参与肿瘤细胞增殖、侵袭、转移以及抑制DNA修复等生物学行为,并诱导机体产生对肿瘤的保护性免疫,是肿瘤诊断与分子靶向治疗中最有应用前景的候选蛋白之一。笔者对TPD52在肿瘤中发生发展的作用及其在肿瘤诊疗中潜在应用价值进行综述,旨在为TPD52进一步研究指明方向,更好实现其临床价值。