Detection Of Cystatin C,IL-4 In Cerebrospinal Fluid And Studies On Immunohistochemistry And Gene In Creutzfeldt-Jakob Disease.

Creutzfeldt-Jakob Disease (CJD), as the most common spongiform encephalopathies, is a group of contagious and inherited neural degeneration disease.Studies show that it is caused by a special transmissible PrP accumulated in the brain.Up to now, the only standard of final diagnosis is biopsy of brain tissue or autopsy to find out PrPsc which is the pathoisomer of PrP.However, biopsy of brain is reluctant to the patients and also easily leads to iatrogenic infection. The main purpose of this dissertation is to find out an effective way to detect early stage CJD marker of cerebrospinal fluid.Proteome studies on CJD patients cerebrospinal fluid show that higher Cys C concentration indicates the damage of the CJD patients brain.Therefore, detecting Cys C is helpful to CJD diagnose. The CJD patients release more pro-inflamatory cytokine than the healthy people. However, there is no research on anti-inflamatory cytokine concentration variation reported. According to my knowledge, this is the first time to detect the Cys C concentration existing in CJD patients cerebrospinal fluid quantitatively.Comparison with other aphrenia is made to make sure whether Cys C detection could be the diagnositic marker of cerebrospinal fluid. Previous studies on the detection of IL-4 concentration shed light on the inflammatory and anti-inflammatory cell's role in the process. Since Cys C and IL-4 are mostly released by gliocyte, observations shows the relationship between activation of microglia and PrP biochemistry types. PRND encoded doppel works in a similar way with PrP and plays an important role in doppel sickness.Since PRND has many point mutations and polymorphism, gene sequencing is made to study the polymorphism on 174 point so as to find its relationship with CJD.In this dissertation,Firstly, two methods, i.e. Western blot and ELISA are used to measure Cys C and IL4 concentration, separately. 39 samples, including 17 CJD, 12 aphrenias and 10 controls are taken into account in this study. The Cys C results is shown as below:For CJD, Cys C concentration varies between 2128 to 8257 ng/ml, with the median 6914 ng/ml which is remarkably higher than aphrenias and controls; For aphrenias, the cys c concentration is also higher than controls.Calculating the cut-off point for the CJD group and the others,best resulus were obtained at a level of 3571ng/ml.At this cut-off point,sensitivity for diagnosis of CJD is 88.2% and specificity is 81.8%.The positive predictive value is 78.9% and the negative predictive value is 90%.Conclusion: The above results indicate that cys c concentration in both CJD and aphrenias increases, but for CJD, the increment is much higher, Hence, the cys c concentration could be taken as the marker of neurolymph of CJD.IL-4 concentration from ELISA:For CJD, IL-4 concentration lies in between 7.413 to 30.881μmol/L, with the median of 9.89μmol/L, which is also much higher than aphrenias and controls. There is no discernible difference between aphrenias and controls. This may be attributed to more microglia activation released by CJD. Most neurolymph of the CJD are gathered in the early stage, therefore, increment of IL-4 concentration indicates that pro-inflamatory cytokines anti-inflamatory cytokines work together in the early stage of CJD.Secondly, chemical observations on microglia and asteroid colloid cell of nine CJDs were made. It leads the following conclusion:more positive CD68 cells are activated by microglia and related to prion deposition as well as biochemistry types. When prion deposits in blocks, microglia activation is obvious while prion deposits in synapse form, there is only a severity activation.These two kinds of activations are both related to prion's biochemistry types and CJD clinical symptom. Activated microglia can release various cell factors, however, increment of positive IL-4 cells is indiscernible, which is most likely caused by cytokine of antiinflammatory release during the early CJD stage contrasting to the sample gathered in the late stage. Positive GFAP cells, which symbolize the astrocyte, increase much but no correlation between activation and prion deposition is observed. Since Cys C, in the brain tissue, are mostly secreted by astrocytes, the increment of positive GFAP cells provide another evidence for the variation of cys c concentration, and further more, cell factors released by activated microglias can promote astrocyte to express cys c. Hence the interaction between activated microglias and astrocytes plays an very important role in the CJD disease.Lastly, PCR method is adopted for PRND gene sequencing on 10 CJDs and RFLP is used to analyze the polymorphism of 174 encoded subpoints.One case of very rare mutation, most likely heredofamilial, is found during PRND gene sequencing. For this case, the 76th base C is replaced by G, which makes the triplet of codon 22 amido acid changed from TCT to TGT, and consequently, the translated protein changes from serine to cysteine (S22C). It's the first time to find S22C in the world. There are contradictory reports on the relationship between PRND 174 situs polymorphism and CJD, so 174 situs polymorphism analysis is conducted in the present study. 12 CJD and 12 healthy persons are chosen. It is found that threonine/methionine heterozygotes appear with a higher frequency in CJDs gene, which implies the linkage between 174 point and CJD. Since the the limitation of our samples, more consecutive work are needed to identify that.