Effects Of Npc1 Gene Mutation On Liver Function And Biological Activity Of Liver-derived Telocytes In Mice

BackgroundNiemann-Pick's disease?NPC1?is a progressively exacerbated disease.Cholecystasis often occurs in the neonatal period,gradually manifesting as hepatosplenomegaly,and the proportion of patients with hepatomegaly is about 31%.Npc1^-/-mice are very similar to human pathogenesis and clinical manifestations.Early hepatosplenomegaly and abnormal liver function,such as alkaline phosphatase and transaminase,are significantly higher than Npc1^+/+ mice.Due to the abnormal aggregation of lipids in liver cells,inflammatory cell infiltration and decreased hepatocyte function lead to the early apoptosis and even fibrosis of liver cells.In recent years,with the deepening of research on Telocytes?TCs?,it has been found to play a key role in maintaining tissue homeostasis and regulating tissue organ development and immune surveillance.At present,the presence of TCs in the liver has been confirmed,but most of the studies on TCs focus on morphological studies,and there are few reports on the morphology and function of TCs in the liver after mutation of Npc1 gene.Therefore,in this study,Npc1^-/-mice?BALB/c Nctr-Npc1m1N/J?were used as experimental animal models to firstly determine the effect of Npc1 gene mutation on liver function and pathological changes in advanced mice,and then detect the morphology of TCs by Npc1 gene mutation.The impact of the study and its multi-directional differentiation potential,the above findings provide theoretical support for further study of the interaction mechanism between TCs biological activity changes and liver dysfunction caused by Npc1 gene mutation.ObjectiveTo clarify the effects of Npc1 gene mutation on liver function and pathological hanges in advanced mice,and further study the effect of Npc1 gene on the biological activity of TCs in liver,and provide theoretical support for further study on the interaction mechanism between TCs biological activity and liver function.Methods1.Identification of genotype of mice by PCR method.2.The activities of lactate dehydrogenase?LDH?,alanine aminotransferase?ALT?and aspartate aminotransferase?AST?in serum were detected by intraocular canthus blood sampling.3.The liver tissues were taken for paraffin and frozen sections.The morphological changes and fat storage of liver tissues were assessed by HE staining and oil red O staining,and the collagen deposition of liver tissues was assessed by Masson staining.4.Real-time PCR and Western blotting were used to detect the expression of proinflammatory factors,interleukin-1beta,interleukin-6 and tumor necrosis factor-alpha in liver tissues.TUNEL staining was used to evaluate the apoptosis of liver tissues.5.The primary TCs of newborn Npc1^+/+ and Npc1^-/-mice were isolatedand cultured by modified type II collagenase digestion method,and the extracted TCs were purified by cell differential adherence method.The growth state of TCs in Npc1^+/+ group and Npc1^-/-group was observed under inverted microscope,and the morphology of TCs under normal growth state was recorded.6.The morphology of TCs was observed by scanning electron microscopy?SEM?,and the cells were identified by transmission electron microscopy?TEM?and immunofluorescence staining.7.Liver TCs of neonatal mice in Npc1^+/+ and Npc1^-/-groups were induced and cultured by adipogenic induction solution,osteogenic induction solution and myocardial induction solution respectively.The induced liver TCs were identified by oil red O staining,alizarin red staining and cellular immunofluorescence.Results1.Gene identification screened newborn Npc1^+/+ and Npc1^-/-mice.2.Compared with the late?P60?Npc1^+/+mice,the weight and liver coefficient of Npc1^-/-mice decreased significantly?P<0.001?,while the activities of LDH,ALT and AST increased significantly?P< 0.001?.3.HE staining showed that the liver tissues of Npc1^-/-mice changed significantly,and a large number of foam cells appeared.Oil red O staining showed that the liver fat content of Npc1^-/-mice was significantly reduced.4.Real-time PCR showed that the expressions of IL-1beta,IL-6 and TNF-alpha in liver tissue of Npc1^-/-mice were significantly increased?P<0.01,P<0.05 and P<0.001?.Meanwhile,Western blotting also showed that the expression of three inflammatory factors were significantly increased?P<0.05,P<0.05 and P<0.01?,which confirmed that the liver of Npc1^-/-mice had late inflammatory reaction;Masson staining did not find the formation of liver fibrosis in Npc1^-/-mice;TUNEL staining showed that the number of apoptotic cells in liver tissue of Npc1^-/-mice increased.5.The results of immunofluorescence staining showed that the TCs cell surface markers Vimentin&CD34,Vimentin&PDGF-a,Vimentin&c-Kit in Npc1^+/+ and Npc1^-/-groups were co-expressed.6.The results of TEM scan showed that the ratio of nucleus/cytoplasm of Npc1^+/+ and Npc1^-/-group TCs was larger than that of other types of cells,and the cell body diameter was 8-16 um.The cytoplasm of cytoplasm in Npc1^+/+ group is very rich,such as microfilament,Golgi,mitochondria,microtubule,intermediate filament,etc.,while the proportion of organelles in cytoplasm of Npc1^-/-group is lower than that of Npc1^+/+ group.7.Under normal microscope and SEM scan,the cell structure of Npc1^+/+ and Npc1^-/-group TCs mainly consisted of cell bodies and protrusions?Tlops?.The cell body of the cell is small,and the cell nucleus is fusiform,star-shaped or spindle-shaped according to the number of Tps.The surface of the TCs cell membrane was uneven,and there was no ifference in morphology between the two groups.8.The results of oil red O staining,alizarin red staining and cellular immunofluorescence showed that the positive rate of TCs after induction was significantly lower than that of Npc1^+/+ group.ConclusionMutation of Npc1 gene causes changes in liver tissue morphology,and a large number of macrophage cells cause inflammation,and the occurrence of inflammatory reaction may be one of the important causes of liver cell apoptosis and impaired liver function;subsequent studies found that liver TCs are in induction conditions.It can differentiate into adipogenic,osteogenic,and cardiomyocyte-like cells;and the Npc1 gene mutation has an inhibitory effect on the multi-directional differentiation potential of TCs in the liver.