| Part I: Isolation,Culture and Characterization of Calcifying Nanoparticles in cystic hepatic hydatidObjective: To isolate and culture Calcifying nanoparticles(CNPs)from the outer capsule wall of cystic hepatic hydatid,and carry out morphological identification to confirm the existence of CNPs in the calcified capsule wall of hepatic hydatid with biliary leakage,so as to provide potential reference for subsequent research.Methods: A total of 37 cases of cystic hepatic hydatid were collected to isolate and culture CNPs from the outer capsule wall tissue of hydatid,with the purpose to observe the relationship between bile leakage of cystic hepatic hydatid and calcification of the outer capsule wall of hydatid.Furthermore,the characteristics of CNPs cultured from the capsule wall were observed under the electron microscope.The expressions of calcification related proteins(OPN and BMP-2)were detected in the outer capsule wall of hepatic hydatid.Further analysis was made focusing on the correlation of bile leakage and CNPs with capsule wall calcification.Results:(1)Quantitative statistical data and CT image analysis confirmed that there was a significant correlation between capsule wall calcification of cystic hepatic hydatid and bile leakage.(2)Immunohistochemistry and Western-blot showed that the expressions of calcification related proteins OPN and BMP-2 in the capsule wall of cystic hepatic hydatid with bile leakage were higher than those in the capsule wall of cystic hepatic hydatid without bile leakage,showing statistically significant difference(P < 0.05).(3)The outer capsule wall,contents,fluid and bile of hydatid were isolated and cultured,and white sediment could be observed at the bottom of the test tube after 4 weeks.The expression of fetuin A was positive by Western-blot,with the highest expression detected in bile.Corresponding findings were consistent with the characteristics of CNPs observed under electron microscope.(4)Quantitative statistical data showed that there was a significant correlation between the presence or absence of CNPs in the outer capsule and the occurrence of biliary fistula,and between the presence or absence of CNPs in the outer capsule wall and the occurrence of biliary fistula and calcification of the outer capsule wall.Conclusions:(1)Our research verifies that hydatid bile leakage is an important factor causing calcification of the outer capsule wall of cystic hepatic hydatid.(2)CNPs are isolated,cultured and confirmed in the calcified outer capsule wall of cystic hepatic hydatid for the first time in this study.(3)This study proves that there is a certain correlation between bile leakage,CNPs and calcification of hepatic hydatid capsule wall,providing an important idea for exploring the natural course of cystic hepatic hydatid and its treatment.Part II: Preliminary Study on Cell Calcification Caused by Calcifying Nanoparticles Acting on Mesenchymal Stem CellsObjective: To explore the mechanism of calcification caused by Calcifying nanoparticles(CNPs)acting on mesenchymal stem cells(MSC),aiming to indirectly confirm the correlation between hepatic hydatid cyst wall calcification and CNPs,so as to provide a basis for clinical research in the future.Methods: The primary cultured mouse MSC were selected and divided in different groups with corresponding treatment adopted in each group as follows: The Control group was given the same amount of normal saline,NHAC group used low-concentration nano-hydroxyapatite-collagen(NHAC),L-CNPs group were supplied with low-concentration CNPs and H-CNPs group was provided with high-concentration CNPs.The cells cultured for 3 and 6 days were detected qualitatively and quantitatively: The morphological changes were observed by light microscope;CCK-8 was used to detect cell proliferation in each group;Apoptosis and calcium concentration were detected by flow cytometry after 6 days;The intracellular calcium deposition was analyzed by alkaline-phosphatase(ALP)staining and alizarin red staining;The expressions of OPN,BMP-2 and TGF-β1/Smad3 proteins were analyzed by immunofluorescence;The expressions of OPN,BMP-2,RUNX2,Bax,Caspase-3,TGF-β1 and Smad3 in cells of each group were detected by Western blot,etc.Results:(1)Under light microscope,the lysis and death of MSC increased significantly with the increase of the culture concentration of CNPs;(2)CCK-8 assay revealed that CNPs could significantly inhibit the proliferation of MSC,showing an enhanced inhibitory effect with the increase of particle concentration and culture time;(3)Flow cytometry indicated that the apoptosis rate was the lowest in the Control group,and that of the NHAC group increased.Moreover,the apoptosis rate increased significantly with the increase of particle concentration;(4)According to the results of alkaline-phosphatase staining,alizarin red staining,flow cytometry detection of calcium concentration and immunofluorescence assay,with the increase of the concentration of CNPs,the expression of ALP,calcium deposition,calcium concentration and the protein expressions of OPN,BMP-2 and TGF-β1/Smad3 in cells increased gradually in a dose-dependent manner;(5)Similar dose-dependent trends were observed according to the results of Western-blot detection of calcification-related proteins BMP-2,OPN and RUNX2,apoptosis-related proteins Bax and Caspse-3,as well as key proteins of TGF-β1/Smad3 signaling pathway.In other words,there existed increased protein expressions with the increase of the concentration of CNPs,and there were statistically significant differences among groups(P < 0.05).Conclusions:(1)As a calcification inducer,CNPs can promote the phenotypic transformation of MSC into osteoblast-like cells by activating TGF-β1/Smad3 signaling pathway,fully inhibit the proliferation and promote the apoptosis of MSC,continuously trigger the disturbance of intracellular calcium metabolism and induce the aggravation of cell calcification in a dose-dependent manner.(2)CNPs have stronger effects on proliferation,apoptosis and calcification of MSC than NHAC. |
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