IR和IGF-1R在糖尿病大鼠拔牙窝愈合过程中的表达及作用
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摘要
糖尿病是严重威胁人类健康的慢性病之一,其中90%以上为2型糖尿病(type 2 diabetes mellitus, T2DM)患者。糖尿病曾是人工种植体的禁忌证,但新的研究表明良好的血糖控制可提高糖尿病患者人工种植牙成功率。目前控制血糖最常用最有效的药物是胰岛素。刘洪臣等提出胰岛素局部给药对成骨细胞有保护作用,并在临床成功应用于糖尿病患者种植体局部获得良好效果。课题组通过体外实验已经证实适量浓度胰岛素影响成骨细胞的增殖和分化,而且发现成骨细胞上有胰岛素受体的表达。胰岛素必须与受体结合才能发挥其生理学效应,受体的数目和与胰岛素的亲和力同胰岛素的生物学效应有直接关系。胰岛素受体(insulin receptor, IR)和胰岛素样生长因子-Ⅰ受体(insulin-like growth factor-I receptor,IGF-IR)属于胰岛素样生长因子系统家族(insulin like growth factors, IGFs)的受体酪氨酸激酶,虽然两者具有高度相似的分子结构和细胞内信号系统,但是IR和IGF-IR在代谢过程、细胞增殖分化中发挥着不同作用,尤其将胰岛素局部应用于糖尿病大鼠拔牙窝后两种受体的表达和变化尚不清楚。Goto-Kakizaki (GK)大鼠是与人类2型糖尿病近似的自发性非肥胖2型糖尿病鼠种,主要表现为葡萄糖刺激的胰岛素分泌受损出现的胰岛素分泌绝对不足。本实验选用适宜生理浓度的胰岛素,利用壳聚糖／β-甘油磷酸钠凝胶(chitosan/β-glycerophosphate, C/GP)系统,将胰岛素溶液凝胶化后局部注射到GK大鼠拔牙窝中,观察IR和IGF-1R的表达与变化以及拔牙窝愈合过程中新生骨组织形成,初步探讨两种受体在糖尿病大鼠拔牙窝愈合过程中的作用。
第一部分胰岛素对下颌骨成骨细胞增殖的影响及IR和IGF-1R在成骨细胞上的表达
目的：探讨适宜浓度对成骨细胞生长增殖的影响,观察IR和IGF-1R在成骨细胞上的表达
方法：原代培养Wistar大鼠下颌骨成骨细胞并鉴定,分别用正常及高糖浓度的培养液配制的不同浓度梯度胰岛素溶液,然后以不同浓度胰岛素液通过培养成骨细胞用噻唑蓝法(MTT法)检测成骨细胞的增殖,确定药物最适浓度；免疫组织化学法检测体外成骨细胞上IR和IGF-1R的表达
结果：通过酶消化法成功培养了原代大鼠下颌骨成骨细胞；在相同浓度胰岛素条件下MTT结果显示成骨细胞在低糖培养基中增殖活跃；在四组不同胰岛素浓度10-6M浓度下成骨细胞增殖最为显著。在体外成骨细胞上IR和IGF-1R均阳性表达。
结论：高糖条件抑制了大鼠下颌骨成骨细胞的增殖；在两种糖浓度下,10-6M胰岛素能显著促进大鼠下颌骨成骨细胞的增殖；而且成骨细胞均表达IR和IGF-1R蛋白。
第二部分胰岛素凝胶缓释系统的制备
目的：用C／GP凝胶系统结合胰岛素制备胰岛素缓释凝胶,并检测胰岛素凝胶效能
方法：高效液相色谱法测定胰岛素局部给药凝胶系统释出胰岛素的量及释放趋势,同时检测胰岛素凝胶系统中β-甘油磷酸钠浓度、pH值对于凝胶系统从水溶液转变为凝胶时间的影响以及释出胰岛素影响
结果：C／GP凝胶系统在25℃下呈液态,37℃时约8min形成凝胶,凝胶形态良好,并且在负载了胰岛素溶液后壳聚糖／β-GP凝胶形态未受影响；GP含量、pH值影响C／GP系统胶凝时间；GP在C／GP系统中终含量为5.6%时胰岛素体外释放较为理想
结论：成功制备了胰岛素壳聚糖／β-甘油磷酸钠凝胶系统,确定了适当的GP含量和pH值范围。
第三部分胰岛素凝胶局部给药促进GK大鼠拔牙窝愈合作用的观察及IR和IGF-1R在拔牙窝愈合过程中的表达及变化
目的：通过组织学、免疫组织化学观察IR和IGF-1R在GK大鼠拔牙窝愈合过程中成骨情况的表达及变化,探讨胰岛素通过两种受体影响糖尿病大鼠拔牙窝愈合的机理
方法：手术拔除大鼠下颌骨左侧切牙；拔牙窝局部注射胰岛素凝胶,设4组：正常大鼠拔牙组(WN)；GK大鼠拔牙组(GN)、GK大鼠无胰岛素C／GP凝胶注射组(G1)、GK大鼠胰岛素C／GP凝胶注射组(G2)；分别在拔牙后7d,14d,21d,28d取材,通过X线观察、组织形态学、免疫组织化学法及qPCR观察IR和IGF-1R在GK大鼠拔牙窝愈合过程中的表达及变化,分析胰岛素结合两种受体与下颌骨拔牙窝愈合的机理。
结果：注射C／GP凝胶于下颌骨拔牙窝后,G2组大鼠血糖检测无统计学差异；免疫组织化学观察IR和IGF-1R均在成骨细胞的胞浆上阳性表达,IGF-1R阳性表达细胞多位于拔牙窝新生骨小梁周围,IR阳性表达的细胞位于拔牙窝内骨陷窝周围；IGF-1R阳性表达的细胞多于IR阳性表达的细胞。HE染色观察在G1,G2组28d时未见有c／GP；Goldner's三色法观察G2组在拔牙后28d拔牙窝内出现不规则骨基质和少许的新生骨小梁,围绕新生骨小梁周围的成骨细胞明显活跃,立方形、多边形的成骨细胞多见。在大鼠拔牙窝7-28d愈合过程中,WN组IR mRNA水平表现为持续下降,GN组则表现为IRmRNA水平在14d达到高峰后开始下降为先升高后降低的趋势,G1组也表现为先升高后降低趋势,21d时达到高峰,随后在28d时降到7d时水平,G2组与WN组表现一致,在7-28d过程中持续下降,其中GN组的IRmRNA水平最高(p<0.01),G1、G2组与WN组相近均低于GN组；此过程中GN、G1组的IGF-1RmRNA水平较低,G2组在7d,14d,21d明显高于其它两组,并在21d时达到峰值,WN组14d,21d显著高于GN、G1组(p<0.01)
结论：胰岛素缓释的凝胶系统局部注射于大鼠拔牙窝内,未即刻改变大鼠血糖；胰岛素局部应用于拔牙窝改变了IR和IGF-1R水平,促进糖尿病大鼠拔牙窝中新生骨形成。
Diabetes is one of the chronic diseases that can cause many serious complications and pose a serious threat to human health. Type 2 diabetes (type 2 diabetes mellitus, T2DM) is by far the most common, affecting 90% of the diabetes population. Diabetes was one of the dental implant contraindications; while good blood glucose control could improve the success rate of dental implants in diabetics. Insulin therapy is a critical part of treatment for diabetics. Professor Liu Hongchen successfully used insulin around the dental implant in diabetics, he proposed that insulin may protect osteoblasts via insulin receptor (IR) and/or insulin-like growth factor 1 receptor. The research team has been confirmed that the effect of insulin on osteoblast proliferation and differentiation, and the expression of IR on osteoblast in vitro.Insulin molecule has docked onto the receptor and effected its action, IR and IGF-IR belongs to the family of tyrosine kinase receptor IGFs, although the IR and the IGF-IR are closely related sharing a high degree of homology, they play a different role in the cell proliferation, differentiation and metabolic process.However, the role of IR and IGF-IR in the healing process of tooth extraction when chitosan-based hydrogel insulin injected is not yet clear. Goto-Kakizaki (GK) rats is a non-obese animal model of spontaneous type 2 diabetes, they are mainly impaired glucose-stimulated insulin secretion. This study utilized the chitosan/β-glycerophosphate hydrogel (chitosan/β-glycerophosphate gels, C/GP) system loaded appropriate physiological concentrations of insulin, injected when the sol to gel in the tooth extraction socket in GK rats. To explore the role of two receptors in the healing process of teeth extraction socket in diabetic rats, expression of two receptors in osteoblasts and new bone formation were observed.
Part I Insulin on osteoblasts proliferation and the expression of IR and IGF-IR in osteoblasts
Objective:To select an appropriate physiological concentration of insulin, and observe the expression of IR and IGF-1R in the osteoblasts
Methods:Isolated, cultured and identified osteoblasts were treated with physiological glucose concentration and high glucose concentration of culture medium prepared with 4 concentrations insulin treated, MTT assay the proliferation of osteoblasts, to determine the appropriate physiological concentration of insulin; identificated expression of IR and IGF-1R of in osteoblasts by immunohistochemistry.
Results:Treated insulin in the same concentration, the proliferation of osteoblasts is more active in L-DMEM; 10-6M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-1R expressed in osteoblasts in vitro.
Conclusion:10-6 M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-IR expressed in osteoblasts in vitro.
PartⅡPreliminary study of chitosan-based hydrogel for the local delivery of insulin
Objective:To establish chitosan-based hydrogel for the local delivery of insulin
Methods:Concentration and rate of insulin released were assayed by HPLC; to detect the gelation time and the amount of insulin released affected byβ-glycerophosphate concentration and pH value.
Results:C/GP gel system was liquid at 25℃, from the sol to gel in 37℃within 8min; the chitosan/β-GP gel systems were not affected after loaded insulin solution; GP content and pH value affect the C/GP gelation time; the final optimal concentration of GP in C/GP system is 5.6%.
Conclusion:Chitosan/β-glycerophosphate gel system has been successfully applied to the local administration of insulin.
PartⅢLocal administration of insulin gel for tooth extraction sockets in GK rats and expression of IR and IGF-1R in the healing process
Objective:To observe the expression of IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and preliminary analyze the relevance of two receptors with new bone in the tooth extraction socket.
Methods:Extraction of the rat incisor; local administration of the chitosan-based hydrogel insulin in tooth extraction socket. Randomized:Wistar group, only the extraction group (WN); GK rats,only the extraction group (GN), tooth extraction with chitosan/GP group (G1), tooth extraction of chitosan/GP contained insulin group (G2). After tooth extraction,7 days,14days,21 days and 28days, rats were sacrifced to remove the mandible quickly. To observe IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and analyze the relevance of two receptors with new bone in the tooth extraction socket.
Result:Immunohistochemical expression of IR and IGF-1R are in the cytoplasm of the osteoblast, IGF-1R positive cells were located around the new bone trabeculae, IR positive cells around the lacunae; the numbers of IGF-1R positive cells are more than those of IR positive cells. HE staining in G1, G2 group at 28 days there was no C/GP; G2 group was observed by Goldner's trichrome at 28 days after tooth extraction that there are some of the new bone formation and irregular bone matrix, active osteoblasts were also found near those area.During the period of tooth socket from 7d to 28d, WN group showed IR mRNA levels continued to decline, GN group showed IRmRNA level began to decrease after reaching a peak at 14d; IRmRNA level in G1 group were the same trend as the GN group, reached the peak at 21 d and, then dropped to the level which same to 7d when in 28d; G2 group consistent with the WN group performance, decreased in the healing process during 7-28d; IRmRNA level in GN group was the highest (p<0.01), toward GN group, IRmRNA level in G1 and G2 groups were lower than those of GN group but close to WN group; In this process, IGF-1R mRNA in GN and G1 group were lower than other groups; IGF-1R mRNA level in G2 group was significantly higher than other groups at 7d,14d,21d, and reached the peak at 21d, WN group were significantly higher than the GN and G1 group atl4d,21d(p<0.01).
Conclusion:Successful injection of the C/GP system contained insulin in the tooth extraction socket in GK rats; local administration of insulin regulates IR mRNA and IGF-1RmRNA level, and new bone formation in G2 group is more active than in GN and G1 group.
第一部分胰岛素对下颌骨成骨细胞增殖的影响及IR和IGF-1R在成骨细胞上的表达
目的：探讨适宜浓度对成骨细胞生长增殖的影响,观察IR和IGF-1R在成骨细胞上的表达
方法：原代培养Wistar大鼠下颌骨成骨细胞并鉴定,分别用正常及高糖浓度的培养液配制的不同浓度梯度胰岛素溶液,然后以不同浓度胰岛素液通过培养成骨细胞用噻唑蓝法(MTT法)检测成骨细胞的增殖,确定药物最适浓度；免疫组织化学法检测体外成骨细胞上IR和IGF-1R的表达
结果：通过酶消化法成功培养了原代大鼠下颌骨成骨细胞；在相同浓度胰岛素条件下MTT结果显示成骨细胞在低糖培养基中增殖活跃；在四组不同胰岛素浓度10-6M浓度下成骨细胞增殖最为显著。在体外成骨细胞上IR和IGF-1R均阳性表达。
结论：高糖条件抑制了大鼠下颌骨成骨细胞的增殖；在两种糖浓度下,10-6M胰岛素能显著促进大鼠下颌骨成骨细胞的增殖；而且成骨细胞均表达IR和IGF-1R蛋白。
第二部分胰岛素凝胶缓释系统的制备
目的：用C／GP凝胶系统结合胰岛素制备胰岛素缓释凝胶,并检测胰岛素凝胶效能
方法：高效液相色谱法测定胰岛素局部给药凝胶系统释出胰岛素的量及释放趋势,同时检测胰岛素凝胶系统中β-甘油磷酸钠浓度、pH值对于凝胶系统从水溶液转变为凝胶时间的影响以及释出胰岛素影响
结果：C／GP凝胶系统在25℃下呈液态,37℃时约8min形成凝胶,凝胶形态良好,并且在负载了胰岛素溶液后壳聚糖／β-GP凝胶形态未受影响；GP含量、pH值影响C／GP系统胶凝时间；GP在C／GP系统中终含量为5.6%时胰岛素体外释放较为理想
结论：成功制备了胰岛素壳聚糖／β-甘油磷酸钠凝胶系统,确定了适当的GP含量和pH值范围。
第三部分胰岛素凝胶局部给药促进GK大鼠拔牙窝愈合作用的观察及IR和IGF-1R在拔牙窝愈合过程中的表达及变化
目的：通过组织学、免疫组织化学观察IR和IGF-1R在GK大鼠拔牙窝愈合过程中成骨情况的表达及变化,探讨胰岛素通过两种受体影响糖尿病大鼠拔牙窝愈合的机理
方法：手术拔除大鼠下颌骨左侧切牙；拔牙窝局部注射胰岛素凝胶,设4组：正常大鼠拔牙组(WN)；GK大鼠拔牙组(GN)、GK大鼠无胰岛素C／GP凝胶注射组(G1)、GK大鼠胰岛素C／GP凝胶注射组(G2)；分别在拔牙后7d,14d,21d,28d取材,通过X线观察、组织形态学、免疫组织化学法及qPCR观察IR和IGF-1R在GK大鼠拔牙窝愈合过程中的表达及变化,分析胰岛素结合两种受体与下颌骨拔牙窝愈合的机理。
结果：注射C／GP凝胶于下颌骨拔牙窝后,G2组大鼠血糖检测无统计学差异；免疫组织化学观察IR和IGF-1R均在成骨细胞的胞浆上阳性表达,IGF-1R阳性表达细胞多位于拔牙窝新生骨小梁周围,IR阳性表达的细胞位于拔牙窝内骨陷窝周围；IGF-1R阳性表达的细胞多于IR阳性表达的细胞。HE染色观察在G1,G2组28d时未见有c／GP；Goldner's三色法观察G2组在拔牙后28d拔牙窝内出现不规则骨基质和少许的新生骨小梁,围绕新生骨小梁周围的成骨细胞明显活跃,立方形、多边形的成骨细胞多见。在大鼠拔牙窝7-28d愈合过程中,WN组IR mRNA水平表现为持续下降,GN组则表现为IRmRNA水平在14d达到高峰后开始下降为先升高后降低的趋势,G1组也表现为先升高后降低趋势,21d时达到高峰,随后在28d时降到7d时水平,G2组与WN组表现一致,在7-28d过程中持续下降,其中GN组的IRmRNA水平最高(p<0.01),G1、G2组与WN组相近均低于GN组；此过程中GN、G1组的IGF-1RmRNA水平较低,G2组在7d,14d,21d明显高于其它两组,并在21d时达到峰值,WN组14d,21d显著高于GN、G1组(p<0.01)
结论：胰岛素缓释的凝胶系统局部注射于大鼠拔牙窝内,未即刻改变大鼠血糖；胰岛素局部应用于拔牙窝改变了IR和IGF-1R水平,促进糖尿病大鼠拔牙窝中新生骨形成。
Diabetes is one of the chronic diseases that can cause many serious complications and pose a serious threat to human health. Type 2 diabetes (type 2 diabetes mellitus, T2DM) is by far the most common, affecting 90% of the diabetes population. Diabetes was one of the dental implant contraindications; while good blood glucose control could improve the success rate of dental implants in diabetics. Insulin therapy is a critical part of treatment for diabetics. Professor Liu Hongchen successfully used insulin around the dental implant in diabetics, he proposed that insulin may protect osteoblasts via insulin receptor (IR) and/or insulin-like growth factor 1 receptor. The research team has been confirmed that the effect of insulin on osteoblast proliferation and differentiation, and the expression of IR on osteoblast in vitro.Insulin molecule has docked onto the receptor and effected its action, IR and IGF-IR belongs to the family of tyrosine kinase receptor IGFs, although the IR and the IGF-IR are closely related sharing a high degree of homology, they play a different role in the cell proliferation, differentiation and metabolic process.However, the role of IR and IGF-IR in the healing process of tooth extraction when chitosan-based hydrogel insulin injected is not yet clear. Goto-Kakizaki (GK) rats is a non-obese animal model of spontaneous type 2 diabetes, they are mainly impaired glucose-stimulated insulin secretion. This study utilized the chitosan/β-glycerophosphate hydrogel (chitosan/β-glycerophosphate gels, C/GP) system loaded appropriate physiological concentrations of insulin, injected when the sol to gel in the tooth extraction socket in GK rats. To explore the role of two receptors in the healing process of teeth extraction socket in diabetic rats, expression of two receptors in osteoblasts and new bone formation were observed.
Part I Insulin on osteoblasts proliferation and the expression of IR and IGF-IR in osteoblasts
Objective:To select an appropriate physiological concentration of insulin, and observe the expression of IR and IGF-1R in the osteoblasts
Methods:Isolated, cultured and identified osteoblasts were treated with physiological glucose concentration and high glucose concentration of culture medium prepared with 4 concentrations insulin treated, MTT assay the proliferation of osteoblasts, to determine the appropriate physiological concentration of insulin; identificated expression of IR and IGF-1R of in osteoblasts by immunohistochemistry.
Results:Treated insulin in the same concentration, the proliferation of osteoblasts is more active in L-DMEM; 10-6M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-1R expressed in osteoblasts in vitro.
Conclusion:10-6 M concentration is the appropriate physiological concentration of insulin for osteoblasts in vitro; IR and IGF-IR expressed in osteoblasts in vitro.
PartⅡPreliminary study of chitosan-based hydrogel for the local delivery of insulin
Objective:To establish chitosan-based hydrogel for the local delivery of insulin
Methods:Concentration and rate of insulin released were assayed by HPLC; to detect the gelation time and the amount of insulin released affected byβ-glycerophosphate concentration and pH value.
Results:C/GP gel system was liquid at 25℃, from the sol to gel in 37℃within 8min; the chitosan/β-GP gel systems were not affected after loaded insulin solution; GP content and pH value affect the C/GP gelation time; the final optimal concentration of GP in C/GP system is 5.6%.
Conclusion:Chitosan/β-glycerophosphate gel system has been successfully applied to the local administration of insulin.
PartⅢLocal administration of insulin gel for tooth extraction sockets in GK rats and expression of IR and IGF-1R in the healing process
Objective:To observe the expression of IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and preliminary analyze the relevance of two receptors with new bone in the tooth extraction socket.
Methods:Extraction of the rat incisor; local administration of the chitosan-based hydrogel insulin in tooth extraction socket. Randomized:Wistar group, only the extraction group (WN); GK rats,only the extraction group (GN), tooth extraction with chitosan/GP group (G1), tooth extraction of chitosan/GP contained insulin group (G2). After tooth extraction,7 days,14days,21 days and 28days, rats were sacrifced to remove the mandible quickly. To observe IR and IGF-1R in the healing process of tooth extraction socket in GK rat, and analyze the relevance of two receptors with new bone in the tooth extraction socket.
Result:Immunohistochemical expression of IR and IGF-1R are in the cytoplasm of the osteoblast, IGF-1R positive cells were located around the new bone trabeculae, IR positive cells around the lacunae; the numbers of IGF-1R positive cells are more than those of IR positive cells. HE staining in G1, G2 group at 28 days there was no C/GP; G2 group was observed by Goldner's trichrome at 28 days after tooth extraction that there are some of the new bone formation and irregular bone matrix, active osteoblasts were also found near those area.During the period of tooth socket from 7d to 28d, WN group showed IR mRNA levels continued to decline, GN group showed IRmRNA level began to decrease after reaching a peak at 14d; IRmRNA level in G1 group were the same trend as the GN group, reached the peak at 21 d and, then dropped to the level which same to 7d when in 28d; G2 group consistent with the WN group performance, decreased in the healing process during 7-28d; IRmRNA level in GN group was the highest (p<0.01), toward GN group, IRmRNA level in G1 and G2 groups were lower than those of GN group but close to WN group; In this process, IGF-1R mRNA in GN and G1 group were lower than other groups; IGF-1R mRNA level in G2 group was significantly higher than other groups at 7d,14d,21d, and reached the peak at 21d, WN group were significantly higher than the GN and G1 group atl4d,21d(p<0.01).
Conclusion:Successful injection of the C/GP system contained insulin in the tooth extraction socket in GK rats; local administration of insulin regulates IR mRNA and IGF-1RmRNA level, and new bone formation in G2 group is more active than in GN and G1 group.
引文
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