1.Screening of nucleic acids affinity to recombinant human TGF-? RⅡ
Lin XIE ; Ren LIU ; Xudong ZHU ; Xiangge HE ; Caiyu CHEN
Journal of Third Military Medical University 2003;0(23):-
Objective To select the aptamer to an extracellular soluble fragment of recombinant human TGF-? receptor Ⅱ (TGF-? RⅡ) in order to antagonize TGF-? effectively by using systematic evolution of ligants by exponential enrichment (SELEX). Methods Initial random RNA library was transcripted in vitro from ssDNA 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-N60-CAGACGACTCCCCGA-3′; rhTGF-? RⅡ was used as target protein. Totally,selection of 8 times was carried out in SELEX experiment. Membrane binding assay was performed to detect the evolution of enriched RNA library; Electrophoretic mobility shift assay (EMSA) was done to determine the affinity between the selected nucleic acid sequence and TGF-? RⅡ. Results Evolution of the enriched RNA library along the increased affinity to TGF-? RⅡ was observed with the development of selection. Two types of dominant sequences were isolated and named as sequence A and sequence B. In membrane binding assay,both sequences A and B showed obvious affinity to TGF-? RⅡ. However,no retarded bands were seen in EMSA. Conclusion The affinity of sequences A and B to TGF-? RⅡ is beyond satisfaction. However,possible sequences with improved affinity to TGF-? RⅡ can be selected by post-SELEX on the basis of candidate sequences A and B.
2.Practice experience of establishment of abdominal heart transplantation model combined with tail vein injection in mice (with video demonstration)
Zhiye BAO ; Jiayi ZHU ; Qian JIAN ; Qi PAN ; Boqian LIU ; Jingxu ZHANG ; Keyi ZHAO ; Caiyu YI ; Hao LIU
Organ Transplantation 2019;10(2):171-
Objective To summarize the practice experience of establishing a stable abdominal heart transplantation model combined with tail vein injection in mice. Methods In the preliminary experiment, 50 pairs of donor and recipient Kunming mice received isotransplantation, 40 pairs of donor and recipient C57BL/6J mice underwent isotransplantation. In the formal experiment, 10 pairs of donor and recipient C57BL/6J mice received isotransplantation, 30 pairs of Balb/c mice as the donor and C57BL/6J mice as the recipient received allotransplantation. The time of each step of the heart transplantation (including harvesting and dressing of the donor heart, vascular anastomosis of the recipient, etc.) was recorded. The duration of transplanted heart beat and the survival time of the recipient was observed daily after operation. The time required for tail vein injection in the transplanted mice was recorded. Pathological examination of the transplanted heart was performed at 30 d after isotransplantation (
3.Effect of Gubitongxiao granules in a mouse model of steroid-induced necrosis of the femoral head
Xiang FANG ; Zhengxin ZHOU ; Lei ZHU ; Ren RUI ; Maoyu XU ; Caiyu ZHU
Chinese Journal of Tissue Engineering Research 2024;28(16):2599-2604
BACKGROUND:Glucocorticoids can inhibit the expression of hub genes in the parathyroid hormone type Ⅰ receptor(PTH1R)/protein kinase A(PKA)signaling axis and interfere with the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells,leading to the disruption of blood supply in bone and bone tissue structures.Previous studies of the research team showed that Gubitongxiao granules can induce blood vessel formation and inhibit osteoblast apoptosis,which has a certain effect on the prevention and treatment of steroid-induced femoral head necrosis. OBJECTIVE:To observe the therapeutic effect of Gubitongxiao granules in a mouse model of steroid-induced femoral head necrosis,and to explore its mechanism from the PTH1R/PKA signaling axis. METHODS:An animal model of steroid-induced necrosis of the femoral head was established by intraperitoneal injection of lipopolysaccharide and gluteal muscle injection of prednisolone acetate.After identification by nuclear magnetic resonance method,60 mice that were successfully modeled were divided into model group,Gubitongxiao granule group and Tongluo Shenggu capsule group,with 20 mice in each group.Another 12 normal mice were used as control group.The corresponding groups were intragastrically given the corresponding drugs for 12 weeks,and then the samples were taken under anesthesia.Histomorphology of femoral head samples was observed by hematoxylin-eosin staining.Enzyme-linked immunosorbent assay was used to detect the serum levels of bone alkaline phosphatase,type Ⅰ amino-terminal extension peptide,parathyroid hormone,osteocalcin and alkaline phosphatase.Western blot and RT-qPCR were used to detect PTH1R,PKA,myocyte enhancer factor 2,sclerostin and guanylate-binding protein activity-stimulating peptide at protein and gene expression levels,respectively. RESULTS AND CONCLUSION:Gubitongxiao granules may reduce the serum PTH level in mice,inhibit the activation of the PTH1R/PKA signal axis,further up-regulate the protein expressions of sclerostin and myocyte enhancer factor 2,and increase the levels of bone alkaline phosphatase,type Ⅰ amino-terminal extension peptide,osteocalcin and alkaline phosphatase in mice,thus improving femoral head necrosis,which is comparable to the intervention effect of Tongluo Shenggu capsules.It is speculated that Gubitongxiao granules may prevent and treating hormonal femoral head necrosis by regulating the PTH1R/PKA signaling axis.