Establishment Of A Method For Measuring Thermal Tolerance Of Serum Complement And Exploration Of Clinical Significance And Molecular Mechanism

Background:Complement is a group of proteins existing in the serum and tissue fluid of normal humans and animals.It can be widely involved in the body’s antimicrobial immune defense response and immune regulation,and is one of the important indicators reflecting the body’s immune function.The activation process of complement is a cascade enzymatic reaction,and it is sensitive to heat.The cascade enzymatic reaction will be affected and the complement’s balance mechanism will make mistakes,when any component of complement changes.Complement abnormalities are closely related to a variety of clinical diseases such as immune diseases,infectious diseases,and tumors.An accurate and stable method for detecting the thermal tolerance of complement was established based on the CH50 hemolysis experiment.And this method was used to measure the complement thermal tolerance of healthy subjects,patients with chronic kidney disease,bone fracture or joint injury and diabetes to prove its clinical application value.The relationship between the polymorphism of HLA-DQB1 gene,telomere length and complement thermal tolerance was discussed,and the factors that affect complement thermal tolerance from the perspective of molecular biology try to be understanded.Methods:1.Human red blood cells was used as a hemolysis indicator system and rabbit-anti-human red blood cell antibody was used as a hemolysin instead of the traditional sheep red blood cells and rabbit-anti-sheep red blood cell antibody in the complement thermal tolerance test.The ratio of complement activity at 47℃ and 37℃was used to calculate the complement thermal tolerance based on the sensitivity of complement to temperature.At the same time,the complement thermal tolerance of different dilutions,different genders,and different age groups was tested to verify the accuracy and stability of the method.2.The angle compared index(ACI)is established by combining the ratio and absolute value.ACI provides a further explanation of the quantitative variations in complex events and evaluation of the risks in different cardinal numbers from new concepts with a common-sense judgment system.It also can be used to quantitatively evaluate the complement thermal tolerance.3.According to the complement thermal tolerance test,the complement thermal tolerance of healthy subjects,patients with chronic kidney disease,bone fracture or joint injury and diabetes is detected.Independent-sample t test was used to compare the difference between case group and control group of samples,which proves that this method has certain clinical application value.4.Polymerase chain reaction was used to analysis HLA-DQB1 genetype,the relationship between complement thermal tolerance and HLA-DQB1 was statistical analysed from a molecular perspective.5.Telomere length was measured by q PCR.The difference analysis were performed on the lower ACI group and the higher ACI group,in order to study the mechanism of complement activity and complement thermal tolerance at the molecular level.Results:1.The traditional sheep red blood cells were replaced by human red blood cells as a hemolysis indicator system,and rabbit-anti-human red blood cell antibody was used as a hemolysin instead of rabbit-anti-sheep red blood cell antibody in the experiment.Because of the sensitivity of complement to temperature,the ratio of complement activity at 47℃ and 37℃ was used to calculate the thermal tolerance of complement on the basis of complement cytolysis and CH50 experiments.A method for measurement complement thermal tolerance was successfully established.It is accurate and stable,and has potential application value.2.ACI,which combines the ratio and absolute value,provides a better assessment method for biomarkers and can be used to evaluate complex events with different cardinal frequency.And it can be used to quantitatively evaluate the complement thermal tolerance.3.The mean value of ACI in healthy subjects was 70.347±6.110,and the mean value of ACI in diabetics was 72.881±5.033.ACI of diabetics was slightly lower than that of healthy subjects(P<0.05).There was a significant difference in distribution between the two groups(P<0.05).The mean value of ACI in patients with CKD was71.319±5.535,and the mean value of CTT in healthy subjects was 73.949±5.673.ACI of patients with CKD was slightly higher than that of healthy subjects(P<0.05).The mean of ACI in patients with bone fracture or joint injury was 70.198±4.326,and the mean of CTT in healthy subjects was 72.122±5.697.There was no significant difference in ACI between the two groups(P>0.05).4.There was no significant difference between the actual observed number and the theoretical number of HLA-DQB1 genotypes,and there was no significant difference in the distribution frequency of HLA-DQB1 alleles between the lower ACI group and the higher ACI group(P > 0.05).5.There was no significant difference in telomere length between the samples of lower ACI and higher ACI groups.There was no significant correlation between ACI and telomere length(P> 0.05).Conclusions:1.A method for detecting complement thermal tolerance was successfully established.2.The detection of complement thermal tolerance can be applied in the process of diagnosis and treatment of diseases,and may be a potential clinical diagnostic value.3.Complement thermal tolerance has different manifestations in different diseases,and the increase or decrease of complement thermal tolerance may be of pathological significance in different diseases.4.There may be no close relationship between ACI and the process of antigen presentation to T cell receptors in the immune response.Complement thermal tolerance may not be affected by biological age and aging at molecular level.