胰岛素瘤动物模型的建立及特性研究

Establishment of insulinoma animal models and observation of tumor characteristics

目的:建立胰岛素瘤的动物模型并分析其特性,为胰岛素瘤的深入研究奠定实验基础。方法:首先检测体外培养的大鼠胰岛素瘤细胞株INS-1的激素释放能力,然后将INS-1细胞移植到裸鼠左肾包膜下,或在移植后18 d或在移植前3 d联合腹腔注射链脲佐菌素(STZ)破坏动物自身胰岛。尾静脉采血检测血糖,当血糖<2.8mmol/L认为胰岛素瘤模型建立。对各种条件建立的模型,观察给予不同刺激物对血糖的影响以及动物血清中胰岛素含量;并对动物胰腺组织和移植细胞的肾脏组织标本进行免疫组化染色检测胰岛素和胰高血糖素。结果:胰岛素瘤细胞既表达胰岛素,同时也表达胰高血糖素。裸鼠接种INS-1细胞后3～4周血糖<2.8 mmol/L;移植肾脏明显增大,形成明显肿瘤,直径≥1 cm。细胞移植后腹腔注射STZ的动物,血糖短暂回升,超过正常血糖水平,之后又逐渐下降,约2周后血糖<2.8 mmol/L。正常裸鼠给予STZ后血糖明显升高,移植INS-1细胞后动物血糖逐渐下降,约4周后血糖降至2.8 mmol/L。与正常对照组相比,3种方法建立的胰岛素瘤模型给予高糖后,动物血糖峰值低。高糖加精氨酸或乙酰胆碱刺激后,正常动物血糖峰值较单纯高糖刺激降低,并较快降至正常水平,但3种胰岛素瘤模型组血糖升高均明显超过单纯高糖刺激者。高糖加去甲肾上腺素刺激后,正常动物血糖达到峰值时间延迟,血糖水平下降缓慢,3种胰岛素瘤模型组血糖较单纯高糖刺激组有所升高。与正常对照组相比,3种胰岛素瘤模型血浆基础胰岛素的水平明显升高。结论:通过给裸鼠肾包膜下移植INS-1细胞,可建立胰岛素瘤动物模型,且瘤细胞同时表达胰岛素和胰高血糖素,不易被STZ破坏。该模型为进一步探讨胰岛素瘤的发病机制奠定实验基础。

AIM： To establish the animal model of insulinoma and to analyze the properties of insulinoma for further study. METHODS： The hormone-releasing ability of rat insulinoma cell line INS-1 was detected in vitro. INS-1 cells were transplanted into the left kidney capsule of nude mice. The islets of the animals were destroyed by intraperitoneal injection of streptozocin （STZ） 3 d before transplantation or 2 weeks after transplantation. The venous blood was sampled, and the level of blood glucose less than 2. 8 mmol/L was defined as successful establishment of insulinoma model. Different irritants were given to the model animals, and the changes of blood glucose and insulin content in serum were observed. The pancreatic tissues and the renal tissues in the injecting sites were taken from all mice for detecting insulin and glucagon by immunohistochemical staining. RF^ULTS： Insulinoma cells expressed insulin and glucagon at the same time. The blood glucose was less than 2. 8 mmol/L 3 to 4 weeks after inoculation of INS-1 cells. Apparent tumor formed in the left kidneys where INS-1 ceils were transplanted and the tumor diameters were more than 1 cm. The level of blood glucose tran- siently increased to higher than the normal level in the mice with tumor cell transplantation after intraperitoneal injection of STZ, and then decreased gradually and returned to less than 2. 8 mmol/L after 2 weeks. The level of blood glucose in the normal nude mice after administration of STZ increased significantly, After transplantation of INS-1 cells, the level of blood glucose decreased gradually, and returned to less than 2. 8 mmol/L after 4 weeks. After stimulated with high glucose, the blood glucose levels in the mice with 3 methods to establish the insulinoma models showed lower glucose peaks than that in the normal control mice. After stimulated with high glucose plus arginine or acetylcholine in the normal animals, the blood glucose peak was lower than that in the normal animals only stimulated with high glucose, and rapidly recovered to the nor- real level. However, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models under the same stimulations were significantly higher than that in the mice only stimulated with high glucose. After stimulated with high glucose plus norepinephrine, the blood glucose peak time in the normal animals delayed, and the blood glucose level declined slowly. After stimulated with high glucose plus norepinephrine, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models increased as compared with that in the mice only stimulated with high glucose. Compared with normal control group, serum insulin in insulinoma mice increased significantly. CONCLUSION： The insu- linoma animal model is successfully established by transplantation of INS-1 cells into the renal capsule of nude mice. The insulinoma cells express both insulin and glucagon, and are not easily damaged by STZ.