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ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.
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ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.
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Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.
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Objective To investigate whether pancreatic islet transplantation in combination with bone mesenchymal stem cells (MSC) transplantation can promote the vascularization surrounding the transplant pancreatic islet.Methods The non-obese diabetic (NOD) mice were utilized as the recipients and randomly divided into pancreatic islet transplantation combined with MSC transplantation group (co-transplantation group,n=6),pancreatic islet transplantation group(n=6),MSC transplantation group(n=6) and sham transplantation group (n=3).The variation in blood glucose level and survival rate post-transplantation of NOD mice in each group was observed.The proliferation and apoptosis of the transplant pancreatic islet in the pancreatic islet transplantation group and co-transplantation group at 1,2 and 4 weeks after pancreatic islet transplantation were analyzed by 5-ethynyl-2'-deoxyufidine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).The vascular density surrounding the transplanted pancreatic islet in the pancreatic islet transplantation group and co-transplantation group at postoperative 2,4 and 8 weeks were observed under light microscope and quantitatively analyzed by histochemical and immunohistochemical staining.Results Both MSC combined with pancreatic islet transplantation and pancreatic islet transplantation significantly improved the blood glucose level and enhanced the survival rate of NOD mouse models after transplantation.In addition,it could accelerate the regeneration of pancreatic islet cells and decrease cell apoptosis.MSC combined with pancreatic islet transplantation significantly enhanced the vascular density surrounding the transplant pancreatic islet compared with pancreatic islet transplantation alone.Conclusions MSC transplantation can accelerate the vascularization surrounding the transplant pancreatic islet,increase the blood supply and protect the function and activity of the transplant pancreatic islet.
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A terminal deoxynucleotidyl transferase ( TdT ) amplification based DNA-copper nanoclusters (CuNCs) sensor was developed for detection of L-histidine ( L-His). Single strand DNA containing poly-thymine ( T) sequences were synthesized by TdT in the presence of dTTP. In blank control, poly-T sequences worked as templates of CuNCs due to the affinity between thymine and copper ions( II) . Fluorescence intensity was enhanced when CuNCs formed with reducing agents. In the presence of L-His, the imidazolyl group of L-His worked as a chelating agent that formed L-His-Cu2+ chelated complex. Thus less copper ions were induced in poly-T sequences, and less CuNCs were obtained to produce week fluorescence signals. A good linear correlation was obtained between fluorescence change and the logarithm of the L-His concentration over the range of 5. 0 ×10-9-5. 0 ×10-4 mol/L. The detection limit was estimated as 3. 4 ×10-9 mol/L. And the recoveries were 97. 4%-104. 6% for the actual urine samples. Compared with other methods of synthetic CuNCs, this method allowed to specifically determining L-histidine without template or labeling, which showed good potential in biomedical and clinical analysis.
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A terminal deoxynucleotidyl transferase ( TdT ) amplification based DNA-copper nanoclusters (CuNCs) sensor was developed for detection of L-histidine ( L-His). Single strand DNA containing poly-thymine ( T) sequences were synthesized by TdT in the presence of dTTP. In blank control, poly-T sequences worked as templates of CuNCs due to the affinity between thymine and copper ions( II) . Fluorescence intensity was enhanced when CuNCs formed with reducing agents. In the presence of L-His, the imidazolyl group of L-His worked as a chelating agent that formed L-His-Cu2+ chelated complex. Thus less copper ions were induced in poly-T sequences, and less CuNCs were obtained to produce week fluorescence signals. A good linear correlation was obtained between fluorescence change and the logarithm of the L-His concentration over the range of 5. 0 ×10-9-5. 0 ×10-4 mol/L. The detection limit was estimated as 3. 4 ×10-9 mol/L. And the recoveries were 97. 4%-104. 6% for the actual urine samples. Compared with other methods of synthetic CuNCs, this method allowed to specifically determining L-histidine without template or labeling, which showed good potential in biomedical and clinical analysis.
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This study aimed to investigate the anti-apoptotic effects of Tangnaikang (TNK) on islet β cells in Zucker diabetic fatty (ZDF) rats.Six male fa/+ ZDF rats were took as the control group,while other thirty male fa/fa ZDF rats were divided into five groups at random:the model group,the metformin group,the high-,mediumand low-dose TNK groups,depending on their body weight and random blood glucose.Prior to the administration,fasting blood glucose and fasting insulin were measured by drawing blood with inner canthusl.Materials were prepared when administered for six weeks.Fasting blood glucose and fasting insulin were detected again.When the sections of the rat pancreatic tissue were embedded,the morphological changes of the islet were observed via HE staining,and the apoptosis of islet β cell were observed using TUNEL.Positive expression of Caspase-3,the transduction enzyme of cell death signal,was tested by immunohistochemical method.It was found that the fasting blood glucose of the (fa/fa) ZDF rats in the high-,medium-and low-dose TNK group was significantly improved after administration (P ＜ 0.01).The serum insulin of rats in the high-,medium-and low-dose TNK group arised compared with the model group,while the high-and low-dose TNK group showed differences in a statistical sense.Compared with the model group,the HOMA-IR of all the treatment group decreased,while significant difference was presented between the high-dose TNK group and the metformin group.HE staining showed that the morphology of the islet β cell of the rats in all the treatment group was improved.The results of TUNEL showed significantly apoptotic changes on islet β cell of the fa/fa ZDF rats.Compared with the model group,the positive expression of TUNEL in the metformin group and the high-dose TNK group were significantly reduced (P ＜ 0.05).The result of immunohistochemistry method showed that the protein levels of Caspase-3 in the metformin group and the high-dose TNK group decreased (P ＜ 0.05).In conclusion,it was demonstrated that TNK effectively reduced the apoptosis of islet β cells in fa/fa ZDF rats,which presented a protective effect.
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Objective To probe the biological effect of multiple intraepithelial injections of recombinant adenovirus-p53(rAd-p53)on inducing the apoptosis in patients with dysplastic oral leukoplakia.Methods 18 patients clinically and histopathologically diagnosed as dysplastic oral leukoplakia were recruited for this study.Intraepithelial injections of rAd-p53 were administered.Immunohistochemistry was used to examine the protein expression of p53 and bcl-2.TUNEL was performed to detect apoptotic cells.Results In the post-treatment patients,p53 protein expression were significantly enhanced(100 ％,18/18),yet bcl-2 protein presented low expression(17 ％,3/18).Statistical analysis revealed the expression of p53 protein had a negative correlation with that of bcl-2 protein(r =-0.837,P ＜ 0.01).15 post-treatment samples (83 ％)were detected obvious apoptotic cells,especially in the samples that were strong p53-positive(r =0.684,P ＜ 0.01).Conclusion Intraepithelial injections of rAd-p53 can induce apoptosis for patients with dysplastic oral leukoplakia.It may be a promising treatment for oral leukoplakia.
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Objective To study the effect of estrogen on myocardial injury in hypothyroidism rats and to reveal the pathology of occurrence and development of the myocardial injury in hypothyroidism rats.Methods The model of hypothyroidism rats was established by feeding propylthiouracil.The pathology of the female group ( n =40 ) and male group ( n =40 ) was observed dynamicly.The presence of myocardial apoptosis cell was demonstrated by the method of terminal deoxynucleotidyl transferase mediated dTUP nick end labeling (TUNEL).And the immunohistochemistry was used to determine the expression of Caspase-3 and Bcl-2 in the myocardial cells.Results The myocardial cell apoptosis appeared in hypothyroidism rats.With the development of the disease,the apoptosis myocardial cell was increased progressively (Female rats group:the numbers of myocardial cell apoptosis at 3,6,8,10,and 12 weeks were 16.40 ± 2.62,29.50 ± 2.67,34.50 ± 3.34,42.70 ±3.24,and 56.00 ± 2.90 respectively,P ＜ 0.05 ; Male rats group:the numbers of myocardial cell apoptosis at 3,6,8,10,and 12 week were 21.60 ± 2.67,35.50 ± 2.94,41.70 ± 2.96,46.70 ± 3.11,and 60.70 ± 3.31respectively,P ＜ 0.05 ),which indicated that estrogen may be related to myocardial cell apoptosis.Conclusion The myocardial cell apoptosis is related with the myocardial injury in hypothyroidism rats.Myocardial cell apoptosis may result in the myocardial injury.The estrogen participates in regulating apoptosis,but its function gradually weakens with the development of the hypothyroidism.
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ymphoblastic leukemia (ALL) of B-cell lineage can be classified using the French- American-British (FAB) classification as L1, L2 and L3 type. L1 and L2 ALLs express terminal deoxynucleotidyl transferase (TdT) and are surface immunoglobulin (sIg)-negative. SIg expression in adults with L1 or L2 ALL is extremely rare. We report a case of L1 ALL with positive sIg. A 39-year-old woman had suffered from fever and abdominal pain for 15 days. Her initial complete blood cell counts were WBC 1.3x109/L, hemoglobin 8.8g/dL and platelet 59.0x109/L. Blast cells on blood were counted up to 24% and showed typical FAB L1 morphology on bone marrow. Immunophenotyping was performed and showed expression of CD5, CD19, CD20, HLA-DR, TdT and sIglamda. Karyotype was 46,XX,der (8;9) (q10;q10),+der (8;9) (q10;q10),t (9;22) (q34;q11.2)[3]/47, idem,+der (22)t (9;22)[5]/46,XX[12]. The case was finally diagnosed as the sIg positive ALL, L1. Chemotherapy consisting of cytoxan, daunorubicin, vincristine, L-asparaginase, prednisolone and intrathecal methotrexate was initiated. The patient had been in complete remission for 12 months. Twelve months later, blasts were detected in cerebrospinal fluid. The patient received intrathecal methotrexate and radiation therapy. Thereafter six months later, blasts were observed on peripheral blood. Bone marrow examination showed diffuse infiltration by blasts with L2 morphology and loss of previously positive sIg. At that time, she had given up the treatment. Although several cases of sIg positive B cell ALL, L1 or L2 have been reported, we could hardly find same case of ours in Korean.
Subject(s)
Adult , Female , Humans , Abdominal Pain , B-Lymphocytes , Blood Cell Count , Blood Platelets , Bone Marrow , Bone Marrow Examination , Cerebrospinal Fluid , Classification , Cyclophosphamide , Daunorubicin , DNA Nucleotidylexotransferase , Drug Therapy , Fever , HLA-DR Antigens , Immunoglobulins , Immunophenotyping , Karyotype , Leukemia , Methotrexate , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Prednisolone , VincristineABSTRACT
OBJECTIVE: Cardiomyopathy, a popular diagnosis that always obscures more than it reveals, nevertheless has several characteristic histological features. These prominently include widespread focal myocardial fibrosis and associated hypertrophy of surviving cardiac myocyte. In fact, focal noninflammatory degeneration (not necrosis) has been demonstrated as a feature of many forms of cardiac hypertrophy. We hypothesized that this loss of myocardial cells in dilated cardiomyopathy (DCMP) may result from cell death by apoptosis. METHODS: Endomyocardial biopsy specimens from the right ventricles of six patients who suffered from DCMP were studied, and myocardial specimens from two persons who died in motor vehicle accidents were used as negative controls. For identification of apoptosis, immunohistochemistry with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling was performed. In addition, apoptosis was confirmed morphologically by confocal laser scanning microscopy with propidium iodide. RESULTS: Apoptosis, that was represented by an apoptotic index ranging from 19.8 to 25.4+ACU-, could be extensively seen in myocytes and also rarely in non-myocytes of interstitium and vascular endothelium. Morphologically, there were a lot of nuclei with clumps of condensed chromatin, suggestive of apoptosis. CONCLUSION: The present study demonstrated that myocyte loss in DCMP might be mainly due to the apoptosis of myocytes and interstitial cells, rather than inflammation or cell necrosis.
Subject(s)
Adult , Female , Humans , Male , Analysis of Variance , Apoptosis , Biopsy, Needle , Cardiomyopathy, Dilated/physiopathology , Cardiomyopathy, Dilated , Caspases/analysis , Enzyme Precursors/analysis , Immunohistochemistry , In Situ Nick-End Labeling , Microscopy, Confocal , Middle Aged , Myocardium , Reference Values , Statistics, NonparametricABSTRACT
In order to explore the significance of cardiomyocyte apoptosis in the early myocardial ischemia,the rat model of acute myocardial ischemia was established,and the apoptotic cells were detected in the early phase of ischemia(within 6h)with TUNEL method.The results showed that scanty apoptotic cells could be observed in the ischemic region 1h after ischemia,and reached the peak 3h after ischemia,then decreased.No apoptotic cells were found in the normal region.In the peri ischemic region,apoptotic cells could also be observed 1h after ischemia,and reached the peak 5h after ischemia.It is indicated that apoptosis is the major form of early ischemic myocardial damage,and the detection of apoptotic cardiomyocyte may provide a new sensitive and objective method for the postmortem diagnosis of early myocardial infarction.
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Objective To study ethanol-induced the neuroapoptosis of visual cortex in offspring mice. Methods Pregnant female mice were fed by intubating alcohol daily,beginning on E5(embryonic,E) and continuing through the pup's birth.The neuroapoptosis in P0,P7 and P14 visual cortex was visualized by Caspase-3 activity immunohistochemistry and Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) staining. Results Usually,the pup's birth days would delay one or two days after ethanol exposure.Moreover,ethanol induced reabsorption of fetus and malformations,such as microcephaly,anencephaly and myeloschisis with spinabifida and so on,were found in the study.Apoptosis index in ethanol treatment groups was obviously higher than that in control at either P0,P7 or P14(P