| The most outstanding characteristics of diabetes is hyperglycemia, which is known as the leading factor of the damage to human health. There are 2 kind of hyperglycemia:continuing hyperglycemia and fluctuant hyperglycemia. Chronic complications of diabetes mellitus are caused by fluctuant hyperglycemia. In order to reduce the risk of chronic complications, research must focus on hyperglycemia fluctuantion of diabetic patient. We should find the pathogenic of the hyperglycemia fluctuantion causing damage to organism and the way of controlling the blood glucose level. Our experiment will establish an animal model of hyperglycemia fluctuantion, investigate the influence to collagenous protein synthesis in kidney and skin, and discuss the mechanism of this influence preliminary. The research have 3 parts.1. To establish the mice model with fluctuant blood glucose:To establish the mice model with fluctuant blood glucose and to compare the influences between non-fluctuant blood glucose groups and fluctuant blood glucose groups. The experimental mice were divided into 4 groups:control group (N), diabetic group (D) and diabetic fluctuant blood glucose group (DF). We established the mice model with fluctuant blood glucose by injecting 0.4mL glucose solution at different time points per day. At the same time, group N and group DF were injected 0.4mL physiclogical salt solution. Detected fasting plasma glucose(FPG) once a week. 8 weeks later, detected average food consumption and water consumption of the mice, and monitor the blood glucose during a day.The experimental result showed that the levels of blood glucose of group DF occurred significant fluctuation in one day. Compared with group N, group D and group DF consumed more food and water. The FPG level of group DF is higher than group D and group N. The daily blood level of group DF is more fluctuated than the other two groups. We can conclude that to inject glucose solution can establish the sensitive, accurate and reliable mice model with fluctuant blood glucose. It can help us to investigate the damage of blood glucose fluctuation to viscera and to find out the relationship with diabetes complications. 2. The influence to collagenous protein synthesis in kidney and primary discuss on the mechanism:Establish the mice model with fluctuant blood glucose, detect the collagen and protein related in kidney, find the cell signaling pathway which has effect on synthesis of collagen protein.8 weeks after the mice model established, we killed the mouse, took their kidney making paraffin section, isolating total protein and mRNA. Type I collagen immunostaining, RT-PCR and western-bloting were used to detecte protein level in kidney.The immunostaining result showed that in renal cortex there are more intercellular space and more protein of type I collagen in group D and DF; in glomerulus, the type I collagen protein level in group DF is higher than that in group D, the level of group N is lowest. The result of RT-PCR and westernbloting showed that the protein level of type I collagen, TIMP1 and Tgf-β1 in group DF is higher than that in group D, the level of group N is lowest; the protein level of MMP1 in group DF is lower than that in group D, the level of group N is highest; the phosphorylation level of ERK smad2 and smad3 in group DF is higher than that in group D, the level of group N is lowest. We can conclude that the blood glucose fluctuation will aggravate the condition of diabetes nephropathy in mice through increase collagen synthesis in kidney, which might be induced by the activation of Tgf-P signaling pathway.3. The influence to collagenous protein synthesis in skin:Establish the mice model with fluctuant blood glucose, detect the collagen and protein related, find the change of collagen protein synthesis in skin.8 weeks after the mice model established, we killed the mouse, took the skin making paraffin section, isolating total protein and mRNA. HE staining, RT-PCR and western-bloting were used to detecte protein level in skin.The HE staining result showed that the skin of group D and DF is thinner, there is no fatty tissue between dermis and musclar layer, and more intercellular space. The result of RT-PCR and westernbloting showed that the protein level of type I collagen, TIMP1 and Tgf-β1 is lower than that in other two groups; the protein level of MMP1 in group DF is higher than that in group D, the level of group N is lowest. We can conclude that the blood glucose fluctuation will aggravate the condition of diabetes dermatopathy in mice through reduce collagen synthesis in skin.Consequently, we can conclude as follows:1. We established a steady blood glucose fluctuation animal model, which will provide a lot help on the research in diabetes and blood fluctuations.2. The blood glucose fluctuation will aggravate the condition of diabetes in mice and damage the kidney more seriously. Blood glucose fluctuation causes much more synthesis of collagen protein. It is the Tgf-βsignal path activation that induced diabetic nephropathy.3. The blood glucose fluctuation will aggravate the condition of diabetes in mice and damage the skin more seriously. Synthesis of collagen protein reduces in the condition of blood glucose fluctuation. |
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