Clinical studyGeneral information

After obtaining the approval of the ethics committee of people's Hospital of Xinjiang Uygur Autonomous Region and the signed informed consent of the children's parents, the cystic fluid (20 ml) and the corresponding paracystal tissues of infected viscera or tissues were collected from children with hydatidosis who met the diagnostic criteria [7] of pediatric hydatidosis in our hospital from January 2020 to June 2021. The paracystal tissues of infected viscera or tissues were used as the control group, and the cystic fluid was used as the case group (n = 40 each). All operations were performed with the consent of the children and their families, and relevant informed documents were signed.

Experimental methods Sample processing

The tissues were washed with pre-cooled phosphate-buffered saline (PBS), cut into pieces, and weighed. The small pieces of tissue were mixed with the corresponding volume of PBS (generally at the weight/volume ratio of 1:9) and fully ground.

Experimental steps

Blank wells (blank control wells without samples and enzyme-labeled reagents) and wells for the sample to be tested were set, respectively. A volume of 40 μl of sample diluent was added into the wells for the sample to be tested on the enzyme-labeled coating plate; next, 10 μl of the sample to be tested was added (the final dilution of the sample was 5×). After sealing the plate with plate sealing membrane, it was incubated at 37°C for 30 minutes. The 30× concentrated washing solution was diluted with distilled water 30× and set aside. The sealing membrane was carefully removed, the liquid was discarded, and the plate was swayed for drying. Each well was filled with detergent, left standing for 30 seconds, and discarded; this procedure was repeated 5×, then the plate was patted dry.

A volume of 50 μL of enzyme-labeled reagent was added to each well, with the exception of the blank well. The plate was incubated at a warm temperature again and washed. Each well was first added with 50 μL of developer A, then with 50 μL of developer B, and was then slightly shaken to mix; the contents then underwent color development at 37°C in the dark for 10 minutes. Each well was added with 50 μL of termination solution, and the reaction was stopped after the blue turned yellow. Zero adjustments were carried out based on the blank well, and the absorbance (optic density) of each well was measured at 450 nm of wavelength according to the number. The measurements were all carried out within 15 minutes after the termination solution was added.

Test indexes

The levels of TGF-β, SMAD2, SMAD3, SMAD4, and LIF in the cystic fluid of children with hydatidosis and the corresponding paracystal tissues of infected viscera or tissues were measured by the enzyme-linked immunosorbent assay (ELISA).

In vitro study In vitro culture and grouping of protoscoleces

The method of Liying Yuan [8] was adopted to digest, separate, and culture protoscoleces obtained from the inner cyst of the hydatid cyst in children with parasitic Echinococcus. The activity of the protoscoleces was detected using 0.1% eosin staining. Protoscoleces with >90% activity were used for testing. Four groups were established for this experiment: the control group (control), the LIF siRNA group (LIF siRNA), the LIF factor group (LIF factor), and the Smad4 siRNA group (SMAD4 siRNA). Each group was triplicate.

Experimental methods

The isolated protoscoleces cultured in a six-well plate were observed. Under the visual field, the number, proportion, and activity of protoscoleces with cystic characteristics were examined and recorded. The protoscoleces were treated with LIF, LIF siRNA interference, and SMAD4 siRNA interference, respectively. After the treatment, the number, proportion, and activity of protoscoleces with cystic characteristics were examined under the visual field and recorded. After microscopic examination, the cultured protoscoleces of each group were killed, and the total protein was extracted. The expression of LIF was detected by ELISA. The correlation between LIF expression after interference and the regulation of protoscoleces cyst formation was statistically analyzed.

Test indexes

Expression levels of TGF-β, SMAD2, SMAD3, SMAD4, and LIF in the samples of Echinococcus Taenia protoscoleces of each group were detected using ELISA. The ELISA method was the same as in the clinical experiment.

Statistical methods

Data were statistically analyzed using the SPSS 17.0 software, and statistical analysis and charting were conducted using the GraphPad Prism 7.00 software. In the comparison of the clinical trial data, measurement data were expressed as mean ± standard deviation (\(\overline\) ± SD), and count data were expressed as percentages (%); comparisons were made using the t-test and rank-sum test, respectively. The data obtained from the in vitro experiment were compared among groups using a one-way analysis of variance (F test). The inspection level was set at α = 0.05.