Erythrocyte Immune Function And CR1 Genomic Polymorphism In Patients With Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (Type 2 DM) is a common, chronic lifelong disease, which seriously damages people's health. Study has discovered that erythrocyte immune function in patients with Type 2 DM is lower, which is connected with the development of the disease process and appearance of its complication. Disagreements exist whether the defective immune function is primary or secondary. Complement receptor 1 (CR1) is the main immune-relating factor on the erythrocyte (red blood cell, RBC) cytomembrane surface, which amount is usually determined by autosomal dominant inheritance and expressed by RBC-CRfs density gene. But for the research of CR1 genomic polymorphism of type 2 DM, nobody has touched both in home and abroad until now. In the present study PCR-RFLP method and RBC adhesion rosetteexperiment with 1:1 case-control study were adopted to observe the RBC immune function and CR1 genomic density polymorphism frequency in patients with type 2 DM, and try to find out the possible mechanism.SubjectsType 2 DM group had fifty-six patients with type 2 DM, including twenty-six male and thirty female, with age of 52.84?5.43 years. Control group had fifty-six healthy persons, including twenty-six male and thirty female too, with age of 53.39+5.22 years, there was no significantly different in sex and age between the two groups (P>0.05). Subjects were drawn 2ml blood before breakfast, 1 ml was put into EDTA-K2 tube and stored in -20癈 refrigerator for ready to extract DNA; Another 1 ml was put into heparin tube and tested the RBC immune function within 4 hours.MethodsI.Test of RBC-CR1 Genomic Polymorphism (1) Thawed Frozen blood samples in room temperature, washed 2 times with sterile double-distilled water, centrifuged 5000 rpm for 5 min and discarded the supernatant, then collected WBC and extracted DNA according to the standardized procedure. (2)The reagents and their amountused in the PCR reaction were: primer 1.0 umol/L, dNTPs 200umol/L, template DNA 3ul, paraffin oil 30ul and buffer solution somewhat. The reaction conditions were as follows: the sample heated at 94 for 240 seconds, and cycled 30 times at 94 for 60 seconds, 55 for 50 seconds, 72 for 50 seconds, followed by a 72 for 300 seconds step. (3) Took amplificated product 5 ul dyed with EB , and electrophoresesed, then placed them under UV lamp to observe if amplification was successful. (4) Added Hind III endonuclease 1ul , BSA 0.2 ul, buffer solution 2 ul and sterling double distilled water 28ml to 14 ul amplified products, then put them into 37 calorstat for 1.5 hours; (5)Took reaction products 10 ul and Marker 5ul respectively, dyed with EB, dot sample in 1% agarose gel and electrophoresis, then observed result under UV lamp, Only which with one band of 1.8kb were HH type, with three bands of 1.8kb, 1.3 kb and 0.5kb were HL type, with two bands of 1.3kb and 0.5 kb were LL type.2. Test of RBC-CR1 rosette and CIC receptor rosette.(1) Washed blood sample 2 times in heparin tube with saline, centrifuged 3000 rpm for 5 min and discarded the supernatant, added saline to the tube and adjusted the amount of erythrocytes to 1.25x107/ml. (2) Dissolved sensitized yeast lyophilized powder and non-sensitized yeast lyophilized powder with saline in the 37 癈 water calorstatrespectively, Washed yeast 2 times with saline, centrifuged SOOOrpm for 5 min and discarded the supernatant, added saline to the tube and adjusted the amount of sensitized yeast or non-sensitized yeast to 1.25x108/ml. (3) Put sample 50 ul into two tubes respectively, added sensitized yeast solution 50ul to tube 1 and non-sensitized yeast solution 50ul to tube 2, mixed completely then put these tubes into 37 water calorstat for 30 minutes. After took them from water, each tube was drop into 200ul saline and 50ul 0.25% glutaraldehyde, snaked them uniformly, took half amount of the solution with sucker and push into pellicle on a slide glass, fixed with methyl alcohol, dyed with Wright's stain then observed under microscope (40x40), counted 200 erythrocytes, whic...