表达人源补体调节蛋白hCD55对异种胰岛移植的保护作用

李欣; 潘登科; 周佳; 闫杰; 陈军; 杜嘉祥; 龚曼琳

doi:10.3969/j.issn.1674-7445.2022.04.010

表达人源补体调节蛋白hCD55对异种胰岛移植的保护作用

doi: 10.3969/j.issn.1674-7445.2022.04.010

610054 成都，电子科技大学医学院（李欣、潘登科、周佳）；成都中科奥格生物科技有限公司（闫杰、杜嘉祥）；四川中科奥格生物科技有限公司（陈军、龚曼琳）

基金项目:

四川省中央引导地方科技发展专项项目 2021ZYD0151

详细信息

作者简介:

通讯作者:
龚曼琳，女，1966年生，硕士，医学生物高级工程师，研究方向为产品开发与质量控制，Email: mlg@clonorgan.com

中图分类号: R617, R587.1
计量
- 文章访问数: 297
- HTML全文浏览量: 100
- PDF下载量: 67
- 被引次数: 0
出版历程
- 收稿日期: 2022-04-06
- 网络出版日期: 2022-07-14
- 刊出日期: 2022-07-15

Protective role of expression of human complement regulatory protein hCD55 in islet xenotransplantation

School of Medicine University of Electronic Science and Technology of China, Chengdu 610054, China

More Information

Corresponding author: Gong Manlin, Email: mlg@clonorgan.com

摘要

摘要: 目的验证猪胰岛细胞上表达人类簇分化抗原55（hCD55）蛋白能否抑制人血清中补体成分的激活。方法选取4只基因型分别为WT（野生型）、GTKO[α-1, 3-半乳糖基转移酶（GGTA1）基因敲除]、GTKO/hCD55和hCD55的成年猪，分离WT、GTKO、GTKO/hCD55猪胰岛细胞，检测其纯度及胰岛素分泌功能，通过琼脂糖凝胶电泳、逆转录聚合酶链反应（RT-PCR）、流式细胞术从DNA、RNA和蛋白水平检测hCD55的表达。在新鲜人血清孵育条件下，进行补体依赖的细胞毒性实验和补体沉积检测。结果分离得到的3种基因型猪胰岛细胞纯度 > 75%，糖刺激指数 > 1。GTKO/hCD55猪胰岛细胞上表达hCD55信使核糖核酸（mRNA）和hCD55蛋白，能够减少人补体C3c和攻膜复合物C5b-9的沉积，降低细胞毒性。结论猪胰岛细胞上表达hCD55蛋白能够抑制人补体激活，降低补体介导的杀伤作用，表明hCD55蛋白在猪胰岛细胞上可发挥补体保护效果，这为hCD55在异种胰岛移植中的应用提供了理论依据。
- 糖尿病 /
- 猪胰岛细胞 /
- 转基因 /
- 人类簇分化抗原55(hCD55) /
- 补体 /
- 立即经血液介导的炎症反应(IBMIR) /
- C3c /
- C5b-9
Abstract: Objective To validate whether the expression of human cluster of differentiation 55 (hCD55) protein in porcine islet cells could inhibit the activation of complement components in human serum. Methods Four adult pigs with WT (wild type), GTKO [α-1, 3-galactosyltransferase (GGTA1) knockout], GTKO/hCD55 and hCD55 genotypes were selected. Islet cells were isolated from WT, GTKO and GTKO/hCD55 pigs, and the purity and insulin secretion function were detected. The expression of hCD55 at the DNA, RNA and protein levels was analyzed by agarose gel electrophoresis, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Complement-dependent cytotoxicity assay and complement deposition assay were performed under the incubation conditions with fresh human serum. Results The purity of isolated porcine islet cells from three genotype pigs was > 75%, and the glycemic index was > 1. The expression of hCD55 messenger RNA(mRNA) and protein in GTKO/hCD55 porcine islet cells decreased the deposition of human complement component C3c and membrane-attacking complex C5b-9, and reduced the cytotoxicity. Conclusions The expression of hCD55 protein in porcine islet cells could inhibit the activation of human complement and reduce complement-mediated killing effect, indicating that hCD55 protein could exert complement protection effect on porcine islet cells. These findings provide theoretical basis for the application of hCD55 in islet xenotransplantation.
- Diabetes mellitus /
- Porcine islet cell /
- Transgene /
- Human cluster of differentiation55(hCD55) /
- Complement /
- Instant blood-mediated inflammatory reaction (IBMIR) /
- C3c /
- C5b-9

HTML全文

下载: 全尺寸图片幻灯片

图 1 3种猪胰岛细胞团的纯度检测

注：A图为WT成年猪胰岛细胞；B图为GTKO成年猪胰岛细胞；C图为GTKO/hCD55成年猪胰岛细胞（DTZ，×100）。

Figure 1. Purity detection of three porcine islet cell mass

下载: 全尺寸图片幻灯片

图 2 hCD55 DNA、RNA及蛋白水平鉴定

注：A图为DNA水平鉴定；B图为RNA水平鉴定；C图为蛋白水平鉴定。

Figure 2. Identification of hCD55 DNA, RNA and protein level

下载: 全尺寸图片幻灯片

图 3 补体依赖的细胞毒性实验

注：与WT比较，^aP < 0.05；与GKTO比较，^bP < 0.05。

Figure 3. Complement dependent cytotoxicity test

下载: 全尺寸图片幻灯片

图 4 猪胰岛细胞上人补体沉积情况

Figure 4. Human complement deposition in pig islet cells

下载: 全尺寸图片幻灯片

表 1 引物序列信息

Table 1. Information of primer sequences

基因	引物序列（5' to 3'）	片段长度（bp）
CD55	上游^①：GATGTACCTAATGCCCAGCCA 下游^①：AGACGGGTAGTACCTGAAGTGG	953
β-actin	上游^①：GACACCAGGGCGTGATGG 下游^①：ACTCCTGCTTGCTGATCCAC	969
CD55	上游^②：AAGGGCAGTCAATGGTCAGA 下游^②：CTCCCGGATTAGGGCATGATT	256
β-actin	上游^②：GGACCTGACCGACTACCTCA 下游^②：ATTCCATGCCCAGGAAGGAG	263
注：①用于DNA的PCR扩增。 ②用于cDNA的PCR扩增。

下载: 导出CSV

参考文献(37)

[1]	PETERSMANN A, MÜLLER-WIELAND D, MÜLLER UA, et al. Definition, classification and diagnosis of diabetes mellitus[J]. Exp Clin Endocrinol Diabetes, 2019, 127(S 1): S1-S7. DOI: 10.1055/a-1018-9078.
[2]	HULL CM, PEAKMAN M, TREE TIM. Regulatory T cell dysfunction in type 1 diabetes: what's broken and how can we fix it?[J]. Diabetologia, 2017, 60(10): 1839-1850. DOI: 10.1007/s00125-017-4377-1.
[3]	ILONEN J, LEMPAINEN J, VEIJOLA R. The heterogeneous pathogenesis of type 1 diabetes mellitus[J]. Nat Rev Endocrinol, 2019, 15(11): 635-650. DOI: 10.1038/s41574-019-0254-y.
[4]	DEDRICK S, SUNDARESH B, HUANG Q, et al. The role of gut microbiota and environmental factors in type 1 diabetes pathogenesis[J]. Front Endocrinol (Lausanne), 2020, 11: 78. DOI: 10.3389/fendo.2020.00078.
[5]	LEE AS, TWIGG SM, FLACK JR. Metabolic syndrome in type 1 diabetes and its association with diabetes complications[J]. Diabet Med, 2021, 38(2): e14376. DOI: 10.1111/dme.14376.
[6]	BELETE R, ATARO Z, ABDU A, et al. Global prevalence of metabolic syndrome among patients with type I diabetes mellitus: a systematic review and Meta-analysis[J]. Diabetol Metab Syndr, 2021, 13(1): 25. DOI: 10.1186/s13098-021-00641-8.
[7]	刘自强, 接传红, 王建伟, 等. 糖尿病视网膜病变免疫机制的研究进展[J]. 眼科新进展, 2022, 42(2): 150-154. https://www.cnki.com.cn/Article/CJFDTOTAL-XKJZ202202013.htm LIU ZQ, JIE CH, WANG JW, et al. Research progress in immune mechanisms against diabetic retinopathy[J]. Recent Adv Ophthalmol, 2022, 42(2): 150-154. https://www.cnki.com.cn/Article/CJFDTOTAL-XKJZ202202013.htm
[8]	杨晓丽, 李明星. 高频超声及弹性成像评估2型糖尿病周围神经病变的临床价值[J]. 实用医学杂志, 2020, 36(3): 390-394. DOI: 10.3969/j.issn.1006-5725.2020.03.023. YANG XL, LI MX. Clinical value of high frequency ultrasound and virtual touch quantification in evaluating peripheral neuropathy of type 2 diabetes mellitus[J]. J Pract Med, 2020, 36(3): 390-394. DOI: 10.3969/j.issn.1006-5725.2020.03.023.
[9]	顾蕙兰, 张梅, 杨涛. 1型糖尿病心血管并发症相关危险因素的研究进展[J]. 中华糖尿病杂志, 2020, 12(12): 1044-1048. DOI: 10.3760/cma.j.cn115791-20200604-00346. GU HL, ZHANG M, YANG T. Research progress of risk factors for cardiovascular complications of type 1 diabetes mellitus[J]. Chin J Diabetes, 2020, 12(12): 1044-1048. DOI: 10.3760/cma.j.cn115791-20200604-00346.
[10]	SOMA D, NIKUMBH T, MANGUS RS, et al. Distal allograft pancreatectomy for graft salvage after pancreas transplantation[J]. Clin Transplant, 2021, 35(6): e14307. DOI: 10.1111/ctr.14307.
[11]	王芳菲, 吕少诚, 贺强. 胰腺移植治疗终末期糖尿病研究进展[J]. 解放军医学院学报, 2020, 41(11): 1118-1121, 1135. DOI: 10.3969/j.issn.2095-5227.2020.11.013. WANG FF, LYU SC, HE Q. Research advances in pancreatic transplantation for end-stage diabetes mellitus[J]. Acad J Chin PLA Med Sch, 2020, 41(11): 1118-1121, 1135. DOI: 10.3969/j.issn.2095-5227.2020.11.013.
[12]	石宜铭. 胰腺移植的现状与展望[J]. 中华消化外科杂志, 2020, 19(12): 1243-1247. DOI: 10.3760/cma.j.cn115610-20200927-00637. SHI YM. Current status and prospect of pancreas transplantation[J]. Chin J Dig Surg, 2020, 19(12): 1243-1247. DOI: 10.3760/cma.j.cn115610-20200927-00637.
[13]	段金良, 白芳, 凌象超, 等. 胰岛移植治疗晚期糖尿病的研究进展[J]. 中华医学杂志, 2021, 101(4): 302-305. DOI: 10.3760/cma.j.cn112137-20200907-02577. DUAN JL, BAI F, LING XC, et al. Research progress of islet transplantation in the treatment of advanced diabetes mellitus[J]. Natl Med J China, 2021, 101(4): 302-305. DOI: 10.3760/cma.j.cn112137-20200907-02577.
[14]	PAN D, LIU T, LEI T, et al. Progress in multiple genetically modified minipigs for xenotransplantation in China[J]. Xenotransplantation, 2019, 26(1): e12492. DOI: 10.1111/xen.12492.
[15]	殷浩. 胰岛移植最新进展及前景展望[J]. 器官移植, 2019, 10(6): 678-683. DOI: 10.3969/j.issn.1674-7445.2019.06.008. YIN H. The latest progress and prospect of islet transplantation[J]. Organ Transplant, 2019, 10(6): 678-683. DOI: 10.3969/j.issn.1674-7445.2019.06.008.
[16]	季峻松, 郭猛. 胰岛移植的历史、现状与挑战[J]. 外科理论与实践, 2019, 24(1): 88-92. DOI: 10.16139/j.1007-9610.2019.01.019. JI JS, GUO M. The history, current situation and challenges of islet transplantation[J]. J Surg Concepts Pract, 2019, 24(1): 88-92. DOI: 10.16139/j.1007-9610.2019.01.019.
[17]	MATSUMOTO S, SHIMODA M. Current situation of clinical islet transplantation from allogeneic toward xenogeneic[J]. J Diabetes, 2020, 12(10): 733-741. DOI: 10.1111/1753-0407.13041.
[18]	王之旭. 异种胰岛移植研究最新进展[D]. 厦门: 厦门大学, 2019.
[19]	VAN DER WINDT DJ, BOTTINO R, CASU A, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets[J]. Am J Transplant, 2009, 9(12): 2716-2726. DOI: 10.1111/j.1600-6143.2009.02850.x.
[20]	COE TM, MARKMANN JF, RICKERT CG. Current status of porcine islet xenotransplantation[J]. Curr Opin Organ Transplant, 2020, 25(5): 449-456. DOI: 10.1097/MOT.0000000000000794.
[21]	EKSER B, COOPER DK. Overcoming the barriers to xenotransplantation: prospects for the future[J]. Expert Rev Clin Immunol, 2010, 6(2): 219-230. DOI: 10.1586/eci.09.81.
[22]	周小青, 刘玉, 唐成程, 等. 敲除GGTA1同时表达人白细胞抗原G5的基因修饰猪的构建[J]. 生物工程学报, 2022, 38(3): 1096-1111. DOI: 10.13345/j.cjb.210655. ZHOU XQ, LIU Y, TAGN CC, et al. Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5[J]. Chin J Biotech, 2022, 38(3): 1096-1111. DOI: 10.13345/j.cjb.210655.
[23]	GARCIA BL, ZWARTHOFF SA, ROOIJAKKERS SH, et al. Novel evasion mechanisms of the classical complement pathway[J]. J Immunol, 2016, 197(6): 2051-2060. DOI: 10.4049/jimmunol.1600863.
[24]	ZEWDE NT, HSU RV, MORIKIS D, et al. Systems biology modeling of the complement system under immune susceptible pathogens[J]. Front Phys, 2021, 9: 603704. DOI: 10.3389/fphy.2021.603704.
[25]	许晅, 李璋, 马群飞, 等. 胞内补体系统的研究进展[J]. 中国免疫学杂志, 2022, 38(4): 482-487. DOI: 10.3969/j.issn.1000-484X.2022.04.019. XU X, LI Z, MA QF, et al. Research progress of intracellular complement system[J]. Chin J Immunol, 2022, 38(4): 482-487. DOI: 10.3969/j.issn.1000-484X.2022.04.019.
[26]	BAKSHI S, CUNNINGHAM F, NICHOLS EM, et al. Mathematical modelling of alternative pathway of complement system[J]. Bull Math Biol, 2020, 82(2): 33. DOI: 10.1007/s11538-020-00708-z.
[27]	LING M, MURALI M. Analysis of the complement system in the clinical immunology laboratory[J]. Clin Lab Med, 2019, 39(4): 579-590. DOI: 10.1016/j.cll.2019.07.006.
[28]	MARTÍNEZ-ALARCÓN L, LIARTE S, QUEREDA JJ, et al. Profiling Human CD55 transgene performance assist in selecting best suited specimens and tissues for swine organ xenotransplantation[J]. Biology (Basel), 2021, 10(8): 747. DOI: 10.3390/biology10080747.
[29]	GELLER A, YAN J. The role of membrane bound complement regulatory proteins in tumor development and cancer immunotherapy[J]. Front Immunol, 2019, 10: 1074. DOI: 10.3389/fimmu.2019.01074.
[30]	FENG H, LI T, DU J, et al. Both natural and induced anti-Sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation[J]. Front Immunol, 2022, 13: 849711. DOI: 10.3389/fimmu.2022.849711.
[31]	DELAUNE V, BERNEY T, LACOTTE S, et al. Intraportal islet transplantation: the impact of the liver microenvironment[J]. Transpl Int, 2017, 30(3): 227-238. DOI: 10.1111/tri.12919.
[32]	容勋精. 人补体调节蛋白CD46与异种移植[J]. 海南医学, 2021, 32(18): 2406-2411. DOI: 10.3969/j.issn.1003-6350.2021.18.026. RONG XJ. Human complement regulatory protein CD46 and xenotransplantation[J]. Hainan Med J, 2021, 32(18): 2406-2411. DOI: 10.3969/j.issn.1003-6350.2021.18.026.
[33]	SCHMIDT P, GOTO M, LE MAUFF B, et al. Adenovirus-mediated expression of human CD55 or CD59 protects adult porcine islets from complement-mediated cell lysis by human serum[J]. Transplantation, 2003, 75(5): 697-702. DOI: 10.1097/01.TP.0000053249.39753.D6.
[34]	张超, 石宁宁, 高景波, 等. 猪Rosa26位点敲入Loxp修饰的hCD55成纤维细胞系的建立[J]. 农业生物技术学报, 2018, 26(6): 1074-1083. DOI: 10.3969/j.issn.1674-7968.2018.06.018. ZHANG C, SHI NN, GAO JB, et al. Establishment of knock-in loxp-modified hCD55 gene fibroblast cell line with porcine (Sus scrofa) Rosa26 locus[J]. J Agric Biotechnol, 2018, 26(6): 1074-1083. DOI: 10.3969/j.issn.1674-7968.2018.06.018.
[35]	谢雨宣, 唐红梅, 李欣, 等. GGTA1基因敲除猪胰岛细胞移植到Ⅰ型糖尿病猕猴3例报道[J]. 中国普外基础与临床杂志, 2021, 28(5): 561-565. DOI: 10.7507/1007-9424.202104047. XIE YX, TANG HM, LI X, et al. Report of 3 cases of transplantation of GGTA1 gene knockout porcine islet cells into type Ⅰ diabetic macaques[J]. Chin J Bases Clin Gen Surg, 2021, 28(5): 561-565. DOI: 10.7507/1007-9424.202104047.
[36]	BRANDHORST H, JOHNSON PR, BRANDHORST D. Pancreatic islets: methods for isolation and purification of juvenile and adult pig islets[J]. Adv Exp Med Biol, 2016, 938: 35-55. DOI: 10.1007/978-3-319-39824-2_4.
[37]	BOTTINO R, BALAMURUGAN AN, SMETANKA C, et al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies[J]. Xenotransplantation, 2007, 14(1): 74-82. DOI: 10.1111/j.1399-3089.2006.00374.x.