Investigation Of The Associate RAPD Molecule Marker For The Syndrome Of Substance Stasis Together With Toxin In Sepsis

1．BackgroundA systemic inflammatory response syndrome triggered by infection is known as sepsis. Sepsis is a serious complication of critical care diseases such as traum, burn, shock, infection, and big operation in clinic, and also the leading cause of sepsis shock and multiple organ dysfunction syndrome. The epidemiological study abrod shows that the fatality of sepsis is more than acute myocardial infarction already; more than 350 thousand people died from the disease every year in Europe and American, the cost reaches up to 250 hundred million U.S. dollars. There are more than 18 million sepsis patients every year , and the number is increasing at 1.5%. The incidence is high and effective therapies is absence, and the cost is expensive. So sepsis is an important disease influencing health, it is worth investigating the pathogenesis and genetic factors and it will be of help in diagnosis and treatment.2．Theory basisThe pathogenesis of sepsis is definitly complicated, it is associated about infection, inflammation, immunization, blood coagulation, tissue damage, and genetic etc. The clinical manifestation and biochemical indicators of sepsis is various. Although the pathogenic microorganism is the same, the clinical manifestation and prognosis maybe completely different. It is associated with environmental agent and disease process, but genetic factor plays an important role in sepsis. It is manifested that whether there is immune response and the degree of respone to infection is inflenced by genetic factors. Study shows that the polymorphism of TNF, Toll like receptors, IL-1, IL-10, CD14, MBL and some other genes may be associate with the affectabilty and mortality of sepsis. These studies may provide theory basis for early recognition, prognosis analysis and gene therapy all others in sepsis.In the field of Chinese medicine and intergration of traditional Chinese and western medicine, there are more deepgoing recognition about sepsis pathogenesis and treatment.According to years of clinical experience, we find that substance stasis and toxin damage and mutually congealed in collaterals extensively in sepsis, and it is different from other diseases which usually considered with substance stasis and toxin mutually congealed in collateral, e.g. acute myocardial infarction and acute cerebral infarction. Also it is different from common infectious disease other than sepsis, e.g. community acquired pneumonia and acute urinary infection, the substance stasis and toxin are limited to definite organs other than extensively. Although there are factors of substance stasis together with toxin in any population, but they are different.The incidence rate, mortality, and treatment costs of sepsis are all very high, so if the genetic fator for syndrome of substance stasis together with toxin in sepsis can be discovered in gene level, it will provide basis for the early diagnosis prevention and cure in traditional Chinese medicine. However, we don't know which gene is associate with substance stasis and toxin, so we choose randomly amplified polymorphic DNA (RAPD) to investigate the polymorphism among different populations.3．MethodCollect cases of sepsis (Population A), acute myocardial infarction (Population B), acute cerebral infarction (Population C), community acquired pneumonia (Population D), and acute urinary infection (Population E), and mix B and C (Population BC) as representative disease for substance stasis together with toxin damaging definite collaterals to compare with sepsis, and mix D and E (Population DE) as representative disease for common infection that substance stasis together with toxin damaging definite organs to compare with sepsis.Collect peripheral blood of patients by EDTA anticoagulation vacuum tube, and subpackag it in 1.5ml EP tubes with 430ul per tube, and freeze them in -86℃for use. Extract genome DNA by Qiagen DNA Mini Kit, measure OD260 by ultraviolet spectrophotometer and calculate DNA concentration, use 54 random primers to proceed RAPD reaction with DNA samples respectively.RAPD reaction system: RAPD cycle:Make 1.5% agarose gel electrophoresis for RAPD production (staining by Gelred), and image with ultraviolet imagery system. Analyse the images, RAPD is dominance marker, and the productions with the same mobility of the same primer are considered as coisogenic, record "1" when exist and "0" when absence, form the bibasil data matrix. Make genetic analysis by POPGENE software, and make fisher's exact test for the distributional difference of every RAPD marker among different populations.4．ResultFrom May 2008 to December 2008, we enrolled 14 patients with sepsis in population A (age from 45 to 81 yrs, average 63.1±13.0 yrs, including 9 male); 7 patients with acute myocardial infarction in population B (age from 51-80 yrs, average 66.9±12.0 yrs, including 5 male), 7 patients with acute cerebral infarction in population C (age from 58-77 yrs, average 69.4±6.6 yrs, including 5 male), i.e. there are 14 patients in population BC(age from 51-80 yrs, average 68.1±9.4 yrs, including 10 male); 7 patients with community acquired pneumonial in population D (age from 18-79 yrs, average 51.1±26.2 yrs, including 3 male) , 3 patients with acute urinary infection in population E (age from 23-45 yrs, average 30.3±12.7 yrs, including 0 male), i.e. there are 10 patient in population DE (age from 18-79 yrs, average 44.9±24.3 yrs, including 3 male). They were patients from emergency ICU or CCU of Dong Zhi Men hospital affiliated to Beijing University of Chinese Medicine, Beijing Friendship Hospital, China-Japan Friendship Hospital, and Beijing Command General Hospital. All of these patients are Han people, and there is no blood relation in and among populations.For the analysis of RAPD results, there are 33 random primers which has ideal amplification in all, and there are 122 ideal markers in all. The analysis of RAPD markers by POPGENE32 software:(1) The Percentage of Polymorphic Loci:There is no difference between the percentages of polymorphic loci in these populations, the percentages are 89.34% in population A, 81.97% in population BC, and 79.51% in population DE. The average percentage is 83.61%. There are 119 polymorphic loci in the whole 38 samples, the whole percentage of polymorphic loci is 97.54%.(2) Genic Variation Statistics:The Nei's gene diversity is 0.3684±0.1626 in population A, 0.3314±0.1874 in population BC, 0.3175±0.1911 in population DE, and the average is 0.3881±0.1396; the Shannon's Information index is 0.5329±0.22023 in population A, 0.4810±0.2582 in population BC, 0.4624±0.2647 in population DE, and the average is 0.5634±0.1771. Although Nei's gene diversity is lower than Shannon's Information index, but they have the same trend, and it shows that the diversity between populations is small.(3) Nei's Analysis of Gene Diversity in Subdivided PopulationsThe general genetic diversity estimated by Nei's gene diversity is Ht＝0.3863, the genetic diversity in populations is Hs＝0.3391, the coefficient of gene differentiation is Gst＝0.1222, i.e. the percentage of genetic diversity between populations is 12.22%, and there are 87.78% in populations. The gene flow calculate by coefficient of gene differentiation is Nm＝3.5908, it shows that there is gene exchange between populations, and prevent genetic differentiation caused by genetic drift.(4) Genetic Identity and Genetic distanceCalculate genetic identity and genetic distance by Nei's gene diversity, the genetic identity is 0.8909 between A and BC, 0.8588 between A and DE, and 0.9288 between BC and DE. The identity between BC and DE is the highest, and the identity between A and DE is the lowest. The genetic distance between A and BC is 0.1155, between A and DE is 0.1522, between BC and DE is 0.0738, the distance between A and DE is the biggest.(5) Dendrogram Based Nei's Genetic DistanceThe dendrogram based on Nei's genetic distance shows that population BC and population DE gather to pop1, then pop1 and population A gather to pop2. The distance between pop1 and BC/DE is 3.69216, the distance between pop1 and pop2 is 3.00130, the distance between pop2 and population A is 6.69345.Analysis of RAPD marker distributional difference among populations by fisher's exact test:In the whole 122 markers, there are 26 RAPD markers which has significant difference between populations (P＜0.05 or P＜0.01). These markers are S08-4, S10-2, S12-4, S13-2, S18-1, S20-4, S23-1, S23-2, S23-3, S25-1, S25-2, S25-3, S25-5, S28-1, S28-2, S28-3, S28-4, S28-5, S37-1, S38-2, S41-1, S43-2, S43-3, S49-1, S49-2, S49-3. And 14 random primers are involved, they are S08(1), S10(1), S12(1), S13(1), S18(1), S20(1), S23(3), S25(4), S28(5), S37(1), S38(1), S41(1), S43(2), S49(3).Among these discrepant RAPD markers, some behaves low occurrence frequency in population A, such as S08-4, S10-2, S12-4, S41-1; some behaves high occurrence frequency in population A, such as S13-2, S18-1, S20-4, S37-1, S38-2. For four random primers, they have more than 2 markers which has significant difference between populations. 2 markers of primer S43 behave high occurrence frequency in population A, and low occurrence frequency in population BC and DE. 3 markers of primer S23 behave high occurrence frequency in population DE, and low or absent in population A and BC. 4 markers of primer S25 behave high occurrence frequency in population A, low occurrence frequency in population BC, especially there is no amplification in population C. 5 markers of primer S28 behave low occurrence frequency in population A. 3 markers of primer S49 behave high occurrence in population BC, then population A, and the lowest in population DE.5．Conclusions (1)It is detected that there is abundant gene polymorphism between individuals and among different disease combined syndrome populations.(2)Genetic analysis shows abundant gene variation in populations, but gene variation between population is low, and there are affluent gene exchanges between populations.(3)Cluster analysis shows that sepsis with substance stasis together with toxin extensively damage and mutually congealed in collaterals syndrome (population A) is different from population BC and population DE, population BC is acute myocardial infarction and acute cerebral infarction which represent syndrome of substance stasis together with toxin damage definite collaterals, population DE is community acquired pneumonial and acute urinary infection which represent common infection also the syndrome of substance stasis together with toxin damage definite organs other than extensively.(4)Fisher's exact test shows that some RAPD markers has significant distributional difference among populations, it prompts that it is possible to find specific molecule markers for the syndrome of substance stasis together with toxin extensively damage and mutually congealed in collaterals in sepsis by method of RAPD.6．Innovation, problem and prospectThe innovation of this study is the use of RAPD DNA molecular markers in studying traditional Chinese medicine syndromes related micro-material base, and in studying the relationship between gene polymorphism and sepsis TCM blood poisoning pathogenesis, and the preliminary study found that RAPD is feasible in studying syndrome related gene polymorphism. And it is possible to find specific molecular markers related to syndrome of substance stasis together with toxin extensively damage and mutually congealed in collaterals in sepsis by method of RAPD.However, it is just a pilot study of material basis related to syndrome of substance stasis together with toxin extensively damage and mutually congealed in collaterals in sepsis by RAPD, the results shows RAPD maybe possible method to find specific molecular markers. The sample in this study is small, and the radom primers is still less, so it is limited to infer the whole by the study and further study is needed. We can make large scale, choose more random primers, or try to improve primer design method by connecting foregone geneome information. Make duplicate test for the molecular markers that worthy further study, both duplicate and test in large samples. In addition, only a firm understanding of molecular marker information may have clinical meaning, so we must retrieve molecular markers, sequencing, and do bioinformatics analysis for interested markers and identify its features. And design specific primer according to sequence information, and transfer it into sequence characterized amplified region (SCAR) marker, and make verification by classical PCR reaction. If it is related to syndrome of substance stasis together with toxin extensively damage and mutually congealed in collaterals in sepsis by various tests, then it maybe worthy in early diagnosis of the syndrome in sepsis.