| Objective To investigate trematode species that infeced the fish of Heng County Guangxi province.Evaluating the identification of metacercariaet effectiveness-by the analysis of morphological combined with ITS2 sequence. Through the experiments on animals infected with Clonorchis sinensis give cancer promoting drug in a short period of time (3～6 months) observate the Clonorchis sinensis carcinogenic and teratogenic effect.Method Small freshwater fishes in a reservoir in Hengxian county were collected. The minced fish was digested by artificial digestion. Metacercarias were collected and classified depend on the morphological characteristics under microscope. The genome DNA of the different morphological metacercaria was extracted, and the ITS gene was PCR-amplified and sequencing. The sequences were blasted in NCBI to find the homologues and built systems phylogenetic tree by Mega6.0.6. The experimental rats were infected with metacercariae of Clonorchis sinensis after extraction of serum on rat tail blood infection in different time period, according to the need of experiment and experimental reagents to serum by enzyme-linked immunosorbent assay (ELISA) kit for detection of cytochrome P450 2E1 in serum (CYP2E1), cytochrome P450 2A6 (CYP2A6), carbohydrate antigen 199 (CA199), alpha fetoprotein (AFP) and lipid peroxidase (Lipo-XY genase LPO) and interleukin 6 (IL-6) od, using standard OD values and the corresponding concentration make standard line, the corresponding serum concentrations can be obtained from each of the corresponding od detection. And the bar chart shows dynamic changes of serum substance; and the data within and between groups were statistically analyzed. On experimental rats twice anatomy, each anatomical half of each group, respectively in 100 days after infection of rats,150 days of anatomy, were taken liver do H-E pathological sections to observe the changes of liver tissue, and the control of two biopsy pathological changes.Results (1) the small fishes in a reservoir in Guangxi Hengxian carrying metacercariae of molecular biology preliminary detection experiments, morphological identification showed that, Heng County, a reservoir fish infection with five different forms of metacercariae; combined with ITS2 sequence analysis showed that, these 5 kinds of 4 different types of Paragonimus metacercaria identification:flukes (Centrocestus formosanus), Japan and crown fluke Holostephanus dubinini Clonorchis sinensis (Clonorchis sinensis) is a hook spine single testosterone trematode (Haplorchis pumilio) in, Taiwan spine, of which there are two different forms of metacercariae molecular identification were encysted metacercaria of Clonorchis sinensis. (2) Clonorchis sinensis infection can cause rat CYP2E1, IL 6-concentration, Clonorchis sinensis infection in rats CA199, AFP, CYP2A6, LPO concentration detected by statistical F test P> 0.05 was not statistically significant. Although the quantitative detection of CA199, AFP, CYP2A6, LPO no significant difference, but it does not explain Clonorchis infection did not affect several of these cytokines may be infected with time has not been reached, the experimental rats were divided two anatomy, anatomy of each respective half of each group in the infected rats were 100 days,150 days, anatomy, liver were removed and do HE pathological changes observed in liver tissue biopsy results showed that can cause infection after Clonorchis edema of liver cells, even slight degeneration and necrosis, and bile duct caused by portal area widened slightly, increasing bile duct, interstitial fibrosis, lymphocyte infiltration scattered, and visible bile duct dysplasia. Has found pre-cancerous changes in liver cirrhosis, liver cancer and bile duct cancer, we can not deny that if the disease continues to develop will likely cause the occurrence of liver cancer.Conclusions (1) detection of four kinds of flukes in Heng fish in a reservoir metacercariae were Clonorchis sinensis, Taiwan spine with flukes, flukes and Haplorchis Japanese crown flukes. Morphological identification alone metacercariae species has some shortcomings, combined with morphological ITS2 sequence analysis can effectively avoid these pitfalls. (2) Clonorchis sinensis infection in rats induced by some cytokines may change, indicating bile duct and liver cell damage, and produce pathological changes. (3) Clonorchis sinensis-infected Rats can cause liver cell swelling, degeneration, slight necrosis, periportal bile duct widened, increasing the small bile ducts, interstitial fibrosis, scattered lymphocyte infiltration. (4) in the United Clonorchis 20mg/LNDMA infection group found bile duct dysplasia phenomenon, which is a pre-cancerous changes in cholangiocarcinoma. |
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