Antifungal Animal Model With Traditional Chinese Medicine And DNA Test In Patients With Hematological Malignancies Complicated By Fungal Infection

Objective:This experiment is performed to investigate the anti-fungal efficacy and clinical significances of traditional Chinese medicine through invasive fungal infections animal model.Methods:12 clean female SD rats were randomly divided into four groups as follows:GroupA:4 rats (immumosuppressive agent(CTX)+spores vaccination+ traditional Chinese medicine treatment);GroupB:4 rats (immumosuppressive agent(CTX)+spores vaccination+control with distilled water);GroupC:2 rats (immumosuppressive agent(CTX)+no spores vaccination);GroupD:2 rats (normal control group, no immune suppression+no spores vaccination). Fungal infection animal model was constructed through intraperitoneal injection of cyclophosphamide,succeeded by Aspergillus spore vaccination(1*107) through nasal instillation. Take blood of the laboratory animals and detect their serum GM(galactomannan testing) regularly. HE staining and PAM staining (Periodic acid methenamine silver staining)of lung tissue for animals in each group, slice the staining tissue for observing under the microscope, to find fungal spores or hyphae. Separate a mount of lung tissues in aseptic conditions for tissue culture.Results:1. GM Testing:with GM testing positive for judging I> 0.5 shows: Immunosuppression+spores vaccination group (groupA+B):GM positive rate is up to 75%, including the rate of group A is 66.7%,and the rate of group B is 81.3%. A, B two groups of comparisons difference was not statistically significant (P> 0.05),with GM testing positive for judging I> 0.7 shows:Immunosuppression+spores vaccination group (groupA+B):GM positive rate is up to 44.4%, including the rate of group A is 8.33%,and the rate of group B is 73.3%. A, B two groups of comparisons difference was statistically significant (P< 0.01). Group C is negative. Group D is negative except a sample which is considered to be polluted.The statistical data of groutp A is lower than group B obviously,there is not obvious statistical significance between them.2. The pathology of lung tissues in fungal infection animal model: Immunosuppression+spores vaccination group (groupA+B):Having seen varying degrees of lung consolidation and inflammatory cell infiltrationin in all of the 8 rats(Picture 1),and Aspergillus hyphae was found in the organizations of two rats in microscopic examination of lung tissue in the 7 days after the vaccination (Picture 2.3). Group C is only immune suppressed, pathologic changes can be seen in the stained lung tissue sections under the microscope. Group D is normal control group, no obvious abnormality has been found in the stained lung tissue sections under the microscope(Picture 4).3. Lung tissue culture:No fungal growth has been found in all the animals' lung tissue after 7 days' culture.Conclusion:1. Traditional Chinese Medicines have therapeutic effect on fungal infected rats.2. GM testing may find the fungal infected lesions before they appearing in the organization, this can be used for early diagnosis of invasive fungal infections. Objective:·Using Real-Time PCR to detect the content of Aspergillus DNA in serum of patients with fever, compare the result to GM test and clinical manifestations, explore the consistency and accuracy.·valuate the early diagnosis of invasive fungal infections value of Real-Time PCR.Methods:Select the malignant tumor patients with fever in Hematology Department of Shandong Provincial Hospital during 2008-2010.Take GM of their serum, using Qiamp DNA Mini Kit to extract Aspergillus DNA from samples of the patients with positive result. Select Aspergillus-specific primers and optimize the experimental methods, establish negative control and the marker, plot the standard curve. Establish SYBR Green Real-Time PCR method for Aspergillus-infected quantitative detection of clinical patients with fever, and compared to the GM Testing, to explore the consistency and accuracy.Results:The concentration and purity of Aspergillus DNA extracted by Qiamp DNA MiNi Kit can meet the needs of Real-Time PCR experiments via equipment testing, but the Real-Time PCR amplification curve shape is dissatisfied, a large number of non-specific amplification can be seen, the correlated ratio of standard curve is 0.97 (<0.99), failed to form an effective one. The PCR products of samples analyzed by agarose gel electrophoresis showed no specific amplification bands, and the standard established for PCR products came out non-specific amplification bands, amplified product size 500Bp. Amplified bands of primer dimer appeared in all samples, size 100bp. All is not the size of the specific 168bp bands we want.Conclusion:The results are not satisfied. After reviewing earnestly experimental process and analyzing the principle we consider that:the experimental design and principle is scientific and reasonable, because of the human factors (including inaccurate, not skillful,and so on) and the abnormal workability of the selected reagent and primers, we fail to achieve the desired results. Due to lack of time, we cannot continue the experiment. The purpose cannot achieve completely.