版纳微型猪1型糖尿病模型的构建方法初探

doi:10.12300/j.issn.1674-5817.2024.060

实验动物与比较医学 ›› 2024, Vol. 44 ›› Issue (4): 384-392.DOI: 10.12300/j.issn.1674-5817.2024.060

版纳微型猪1型糖尿病模型的构建方法初探

刘凯¹^,²(), 胡毓琦¹^,³(), 耿亚田¹^,², 程文杰¹^,², 王晶¹^,², 魏太云¹, 赵红芳¹^,³, 李加宇¹^,³, 角德灵¹^,³, 赵红业¹^,³(), 魏红江¹^,²^,³()()

^1.云南省小型猪基因编辑与异种器官移植重点实验室, 昆明 650201
^2.云南农业大学动物科学技术学院, 昆明 650201
^3.云南农业大学动物医学院, 昆明 650201

收稿日期:2024-04-22 修回日期:2024-07-06 出版日期:2024-09-06 发布日期:2024-08-25
通讯作者: 魏红江（1971—），男，博士，教授，博士生导师，主要从事动物基因编辑与体细胞克隆研究。E-mail： hongjiangwei@126.com。ORCID： 0000-0002-5663-1093
赵红业（1974—），女，博士，教授，博士生导师，主要从事疾病动物模型构建和分子肿瘤学研究。E-mail： hyzhao2000@126.com。
作者简介:刘凯（1995—），男，博士研究生，研究方向：医用供体猪研发与实验用小型猪疾病检测。E-mail： 1273461372@qq.com；
胡毓琦（1998—），女，硕士研究生，研究方向：医用供体猪研发与实验用小型猪疾病检测。E-mail： 1710693582@qq.com；
耿亚田（1997—），女，硕士研究生，研究方向：动物遗传育种与繁殖。E-mail: 4839313797@qq.com
赵红业，博士，二级教授，博士生导师，现就职于云南农业大学动物医学院，兼任云南省实验动物学会理事。主要从事异种移植用供体猪开发、人类重大疾病基因编辑小型猪模型创制和抗癌药老药新用机制等方面的相关研究。主持国家自然科学基金面上项目及地区项目各1项，与江西农业大学、浙江大学滨江研究院、杭州启函生物科技有限公司等高校、研究院和企业共签订合作研发合同30余份。先后开发了多种基因编辑克隆猪，为异种器官移植、疾病模型、分子标记育种等领域的研究提供了素材，并在Science、Nature Biomedical Engineering、Cancer Research等刊物上发表学术论文60余篇。以第二完成人获云南省技术发明一等奖1项，制定地方标准1部，以第一发明人获得授权发明专利1项，以参与发明人获得授权发明专利9项和实用新型专利2项。E-mail： @126.com。ORCID： 0000-0002-5900-3033
魏红江，博士，二级教授，博士生导师，云南农业大学副校长。系国家重点研发计划首席科学家，全国兽医专业学位研究生教育指导委员会委员，云南省“兴滇英才支持计划”科技领军人才，云南省高层次创新创业团队带头人，动物基因编辑与体细胞克隆技术云南省创新团队带头人，云南省小型猪基因编辑与异种器官移植重点实验室主任，云南省异种器官移植工程研究中心主任。长期从事猪基因编辑、体细胞克隆、异种器官移植、疾病模型创制及实验动物化等研究，获得了世界上首批内源性逆转录病毒失活克隆猪、首例孤雌生殖克隆猪，成功构建了10余种多基因编辑异种器官移植供体猪和基因编辑猪疾病模型。主持国家重点研发计划、云南省重大科技专项计划等国家级、省部级科研项目40余项。以第一作者（含并列）/通信作者（含共同）身份在Science和Nature Biomedical Engineering等知名刊物发表SCI论文50篇。以第一发明人获得授权发明专利9项和实用新型专利2项。获批云南省地方标准2项。以第一完成人获云南省技术发明一等奖1项，其他省部级科技奖3项。E-mail： hongjiangwei@126.com。ORCID： 0000-0002-5663-1093
第一联系人：这3位的贡献同等，为共同第一作者
基金资助:
云南省重大科技专项“人类重大疾病小型猪模型的构建及平台建设”(202102AA100054)

Preliminary Study on Construction Method of Type 1 Diabetes Model in Banna Miniature Pig

LIU Kai¹^,²(), HU Yuqi¹^,³(), GENG Yatian¹^,², CHENG Wenjie¹^,², WANG Jing¹^,², WEI Taiyun¹, ZHAO Hongfang¹^,³, LI Jiayu¹^,³, JIAO Deling¹^,³, ZHAO Hongye¹^,³(), WEI Hongjiang¹^,²^,³()()

^1.Key Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, China
^2.Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
^3.College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Received:2024-04-22 Revised:2024-07-06 Published:2024-08-25 Online:2024-09-06
Contact: WEI Hongjiang (ORCID: 0000-0002-5663-1093), E-mail: hongjiangwei@126.com;
ZHAO Hongye （ORCID: 0000-0002-5900-3033）, E-mail: hyzhao2000@126.com;

摘要/Abstract

摘要：

目的构建1型糖尿病微型猪模型，并探索有效延长模型猪生存周期的术后护理方案。方法选取7头版纳微型猪进行胰腺切除手术。术后3～5 d通过补充葡萄糖、维生素及抗生素等方法帮助其恢复。每日早晚两次检测血糖、尿糖水平，据此调整胰岛素补充量。每日巡视模型猪并记录其饮食欲、精神状态、虚弱程度、皮肤破损情况、粪尿量等数据，每周称量体重，直至动物死亡。根据模型猪状态及时给予葡萄糖注射液和乳酸林格氏液等以补充营养并纠正电解质失衡。结果 7头版纳微型猪在胰腺切除术后均表现为随机血糖浓度大于11.1 mmol/L，远超正常猪的平均血糖值（6.0 mmol/L），尿糖阳性，且其体重呈渐进性下降等典型的糖尿病临床表征，提示1型糖尿病模型构建成功。此外，存活时间超过8周的1型糖尿病猪还出现了渐进性被毛脱落及皮肤破溃的情况；在模型猪无法站立乃至无法自主进食时进行安乐死，并采集其肝脏、肾脏及皮肤等受累及的器官组织进行病理组织切片及HE染色，病理组织切片也可见长期高血糖导致的肝脏淤血、肝糖原大量蓄积、肝细胞气球样变和渐进性肝脏纤维化，肾小球淤血、肾小管上皮细胞空泡变性和蛋白尿，真皮层出现淤血、血管壁变薄以及不同程度的角化不全和角化不良等肝脏、肾脏及皮肤组织病变。本研究构建的版纳微型猪糖尿病模型平均生存期为44 d，最长可生存121 d。结论通过胰腺切除术可成功建立版纳微型猪的1型糖尿病模型，进一步通过术后精心护理可获得并发症明显的长期1型糖尿病微型猪模型，这为1型糖尿病治疗策略研究提供了稳定的大动物模型。

关键词: 版纳微型猪, 1型糖尿病模型, 胰腺切除术, 术后护理, 糖尿病并发症

Abstract:

Objective To construct a Type 1 diabetes model in miniature pigs and explore postoperative care strategies for effectively prolonging the survival time of the model pigs. Methods Seven Banna miniature pigs were selected for pancreatectomy. Glucose, vitamins, and antibiotics were administered for 3-5 days after surgery to aid recovery. Blood glucose and urine glucose levels were measured twice a day in the morning and evening to adjust insulin supplementation accordingly. The model pigs were observed daily and records were kept, including orexis, psychosis, weakness, skin ulcer, and feces and urine. Body weight was measured weekly until the death of the model animals. Based on the model pigs' condition, glucose injection and Ringer's lactate solution were administered to supplement nutrition and correct electrolyte imbalances. Results All seven Banna miniature pigs showed typical symptoms of diabetes: random blood glucose levels higher than 11.1 mmol/L after pancreatectomy, far exceeding the average blood glucose level of 6.0 mmol/L in normal pigs; positive urine glucose; and progressive weight loss. These features indicated the successful construction of Type 1 diabetes model. Additionally, Type 1 diabetic pigs that survived more than 8 weeks showed progressive hair loss and skin ulceration. Euthanasia was performed on model pigs when they were unable to stand or even eat independently, and pathological examination and HE staining were conducted on tissues collected from affected organs such as the liver, kidneys, and skin. Pathological sections revealed liver congestion, massive glycogen accumulation, ballooning degeneration of hepatocytes, and progressive liver fibrosis, along with glomerular congestion, vacuolar degeneration in renal tubular epithelial cells, proteinuria, dermal congestion, thinning of vascular walls, and varying degrees of parakeratosis and dyskeratosis in the liver, kidneys, and skin tissues due to prolonged hyperglycemia. The average survival time of the constructed Banna miniature pig diabetes model was 44 d, with a maximum survival time of 121 d. Conclusion Type 1 diabetes model can be constructed successfully in Banna miniature pigs through pancreatectomy. With meticulous postoperative care, a long-term Type 1 diabetes model with significant complications can be achieved, providing a stable large-animal model for Type 1 diabetes treatment strategies.

Key words: Banna miniature pig, Type 1 diabetes model, Pancreatectomy, Postoperative care, Complications of diabetes

中图分类号:

R587.1

刘凯,胡毓琦,耿亚田,等. 版纳微型猪1型糖尿病模型的构建方法初探[J]. 实验动物与比较医学, 2024, 44(4): 384-392. DOI: 10.12300/j.issn.1674-5817.2024.060.

LIU Kai,HU Yuqi,GENG Yatian,et al. Preliminary Study on Construction Method of Type 1 Diabetes Model in Banna Miniature Pig[J]. Laboratory Animal and Comparative Medicine, 2024, 44(4): 384-392. DOI: 10.12300/j.issn.1674-5817.2024.060.

图/表 6

表1 版纳微型猪胰腺切除术后状态评分表

Table 1 Scoring table for post-pancreatectomy status of Banna miniature pigs

观察指标 Observation indicators	评分 Score
观察指标 Observation indicators	0	1	2	3	4	5
饮食欲 Orexis	饲料无采食痕迹，拒食	饲料有采食痕迹，但未见明显减少	饲料有采食痕迹且减少约1/3	饲料有采食痕迹且减少约2/3	料槽无剩余饲料，加料时有采食意愿但不强烈	料槽无剩余饲料，且加料后采食意愿强烈
精神状态 Psychosis	精神沉郁，目光呆滞无神，对外界刺激无明显反应，不愿与人互动	精神较沉郁，对外界刺激有轻微反应，互动时反应迟钝	精神较沉郁，对外界刺激有一定的反应，互动时反应稍迟钝	精神较正常，对外界刺激有正常的反应，互动时反应速度正常	精神较亢奋，对外界刺激反应较激烈，互动时反应较灵敏	精神亢奋，对外界刺激敏感，互动时反应积极迅速
虚弱程度 Weakness	活动意愿强烈，趴卧较少，无需驱赶或引诱即可观察到站立活动	除采食等生理活动外，有一定的活动意愿，驱赶或引诱时可迅速站立，无需辅助站立	除采食等生理行为外，还具有一定的活动意愿，驱赶或引诱时可站立，无需辅助站立	较喜卧，除采食等生理需求外不愿站立活动，驱赶或引诱时可自主站立移动，但仍表现为不愿站立，辅助站立后可支撑并正常行走，后自行趴卧	喜卧，驱赶或引诱时不愿站立，辅助站立后可勉强支撑，后摔倒	喜卧，驱赶或引诱均不站立，辅助站立后立即摔倒，无法自主维持站立
皮肤破损情况 Skin ulcer	皮肤未见破损	皮肤破损面积约10%	皮肤破损面积约20%	皮肤破损面积约30%	皮肤破损面积约40%	皮肤破损面积约50%及以上
粪尿量 Feces and urine	笼具中未见粪便、尿液残留，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见极少量粪便、尿液，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见少量粪便、尿液，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见一定量粪便、尿液，偶尔能观察到排便、排尿	笼具中见较多量粪便、尿液，经常能观察到排便、排尿	笼具中见大量粪便、尿液，总是能观察到排便、排尿

表1 版纳微型猪胰腺切除术后状态评分表

Table 1 Scoring table for post-pancreatectomy status of Banna miniature pigs

观察指标 Observation indicators	评分 Score
观察指标 Observation indicators	0	1	2	3	4	5
饮食欲 Orexis	饲料无采食痕迹，拒食	饲料有采食痕迹，但未见明显减少	饲料有采食痕迹且减少约1/3	饲料有采食痕迹且减少约2/3	料槽无剩余饲料，加料时有采食意愿但不强烈	料槽无剩余饲料，且加料后采食意愿强烈
精神状态 Psychosis	精神沉郁，目光呆滞无神，对外界刺激无明显反应，不愿与人互动	精神较沉郁，对外界刺激有轻微反应，互动时反应迟钝	精神较沉郁，对外界刺激有一定的反应，互动时反应稍迟钝	精神较正常，对外界刺激有正常的反应，互动时反应速度正常	精神较亢奋，对外界刺激反应较激烈，互动时反应较灵敏	精神亢奋，对外界刺激敏感，互动时反应积极迅速
虚弱程度 Weakness	活动意愿强烈，趴卧较少，无需驱赶或引诱即可观察到站立活动	除采食等生理活动外，有一定的活动意愿，驱赶或引诱时可迅速站立，无需辅助站立	除采食等生理行为外，还具有一定的活动意愿，驱赶或引诱时可站立，无需辅助站立	较喜卧，除采食等生理需求外不愿站立活动，驱赶或引诱时可自主站立移动，但仍表现为不愿站立，辅助站立后可支撑并正常行走，后自行趴卧	喜卧，驱赶或引诱时不愿站立，辅助站立后可勉强支撑，后摔倒	喜卧，驱赶或引诱均不站立，辅助站立后立即摔倒，无法自主维持站立
皮肤破损情况 Skin ulcer	皮肤未见破损	皮肤破损面积约10%	皮肤破损面积约20%	皮肤破损面积约30%	皮肤破损面积约40%	皮肤破损面积约50%及以上
粪尿量 Feces and urine	笼具中未见粪便、尿液残留，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见极少量粪便、尿液，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见少量粪便、尿液，且未观察到膀胱有尿液蓄积或包皮憩室充盈	笼具中见一定量粪便、尿液，偶尔能观察到排便、排尿	笼具中见较多量粪便、尿液，经常能观察到排便、排尿	笼具中见大量粪便、尿液，总是能观察到排便、排尿

图1 胰腺切除术后版纳微型猪的血糖（A）和尿糖（B）检测结果注：Pig 1，Pig 2分别为1、2号胰腺切除术后模型猪，每个点均代表一次检测，7周、11周时观察到实验动物状态异常，并给药进行支持治疗，过程中难以采集符合测定要求的血液，好转后继续监测。结果显示，胰腺切除术后模型猪的血糖浓度绝大多数时间均远超正常范围（橘色细密虚线为对照组猪血糖浓度平均线6.0 mmol/L，红色粗稀线为随机血糖浓度的阈值线11.1 mmol/L），提示糖尿病模型构建成功。

Figure 1 Results of blood glucose (A) and urine glucose (B) in Banna miniature pigs after pancreatectomyNote：Pig 1 and Pig 2 refer to the post-pancreatectomy model pigs numbered 1 and 2, respectively. Each point represents one measurement. At 7 weeks and 11 weeks, abnormal conditions were observed in the experimental animals, and supportive treatment was provided. It was difficult to collect blood samples that met the requirements of the measurement during this time, but monitoring continued once they improved. The results showed that the blood glucose concentration in the model pigs after pancreatectomy was far beyond the normal range for most of the time (the orange fine dashed line represents the mean blood glucose concentration of pigs in control group at 6.0 mmol/L, while the red thick sparse line represents the threshold for random blood glucose concentration at 11.1 mmol/L). This suggested that the diabetic model was successfully constructed.

图2 胰腺切除术后版纳微型猪的存活时间注：A，7头版纳微型猪经胰腺切除术后的存活时间，图中数字为具体生存天数；B，胰腺切除术后护理时给是否予维生素补充治疗的两组版纳微型猪生存曲线比较。

Figure 2 Survival time of Banna miniature pigs after pancreatectomyNote：A, Survival time of 7 Banna miniature pigs after pancreatectomy, with the numbers in the figure indicating the specific survival days; B, Survival curves of two groups of Banna miniature pigs during post-pancreatectomy operative care, comparing those that received vitamin supplementation with those that did not.

图3 胰腺切除手术后不同时间版纳微型猪的外观变化注：A，1号猪术后第1周；B，1号猪术后第4周；C，1号猪术后第15周。

Figure 3 Appearance changes of Banna miniature pigs at different times after pancreatectomyNote：A, One week post-operation for pig 1; B, Four weeks post-operation for pig 1; C, Fifteen weeks post-operation for pig 1.

图4 胰腺切除术后版纳微型猪的体重及状态评分变化注：A，体重变化；B，食欲评分；C，皮肤破损情况；D，粪尿量评分；E，虚弱程度；F，精神状态。其中，A图显示1、2号模型猪，B～F图显示1号模型猪，红色线为各指标趋势线。

Figure 4 Changes in body weight and status scores of Banna miniature pigs after pancreatectomyNote： A, Changes in body weight; B, Scores of orexis; C, Scores of skin ulcer; D, Scores of feces and urine; E, Scores of weakness; F, Scores of psychosis. Figure A shows model pig 1 and 2, while figure B-F show model pig 1. The red line represents the trend line of each indicator.

图5 版纳微型猪胰腺切除术后肝、肾和皮肤组织剖检大体观察及HE染色结果（×400）注：1、2号猪为腺切除术后存活时间最长的糖尿病版纳微型猪，对照猪为未经手术及任何处理。标识R指示红细胞，F指示纤维化，G指示糖原，Pu指示蛋白尿，Pk指示角化不全。

Figure 5 Gross examination and HE staining results (×400) of liver, kidney, and skin tissues in Banna miniature pigs after pancreatectomyNote：Pig 1 and 2 were diabetic Banna miniature pigs with the longest survival time after pancreatectomy. Pigs in the control group were not subjected to operation or any treatment. R, Red blood cells; F, Fibrosis; G, Glycogen; Pu, Proteinuria; Pk, Parakeratosis.

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版纳微型猪1型糖尿病模型的构建方法初探

Preliminary Study on Construction Method of Type 1 Diabetes Model in Banna Miniature Pig

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