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1.
Article in Chinese | WPRIM | ID: wpr-1003786

ABSTRACT

Parkinson's disease (PD) is a kind of chronic progressive neurodegenerative disease that has a high prevalence rate in recent years, especially in the elderly. PD belongs to the category of "tremor disease" and "tremor" in traditional Chinese medicine, and Tianma Goutengyin is a classic prescription contained in the Synopsis of The New Significance of Patterns and Treatment in Miscellaneous Diseases(《中医内科杂病证治新义》). This article explored the theory of Tianma Goutengyin in the treatment of PD, and based on network pharmacological research, the article summarized relevant research on Tianma Goutengyin and its single herb in the treatment of PD. Moreover, it discussed the clinical applications and mechanisms of Tianma Goutengyin and its single herb in the treatment of PD. It is found that the mechanisms of Tianma Goutengyin in treating PD may be related to resisting oxidative stress, inhibiting inflammatory response, regulating neurotransmitters, and protecting dopamine (DA) neurons. Besides, the main components of the single herb in Tianma Goutengyin for treating PD are gastrodin, rhynchophylline, geniposide, gardenial alcohol, eucommitol glycoside, motherwort alkaloid, baicalin, pachyman, and achyranthes bidentata sterol. They can improve the related symptoms of PD patients by inhibiting inflammatory response, resisting oxidative stress, affecting calcium ion concentration, restoring mitochondrial function, and and protecting DA neurons. This article summarized the research progress of Tianma Goutengyin and its single herb in treating PD, so as to provide a reference for the prescription and medication of Tianma Goutengyin in the treatment of PD and subsequent research on the mechanisms of Tianma Goutengyin and its single herb in the treatment of PD and give play to the advantages of traditional Chinese medicine in the treatment of PD.

2.
Acta Pharmaceutica Sinica B ; (6): 4823-4839, 2023.
Article in English | WPRIM | ID: wpr-1011207

ABSTRACT

Clinical application of doxorubicin (DOX) is heavily hindered by DOX cardiotoxicity. Several theories were postulated for DOX cardiotoxicity including DNA damage and DNA damage response (DDR), although the mechanism(s) involved remains to be elucidated. This study evaluated the potential role of TBC domain family member 15 (TBC1D15) in DOX cardiotoxicity. Tamoxifen-induced cardiac-specific Tbc1d15 knockout (Tbc1d15CKO) or Tbc1d15 knockin (Tbc1d15CKI) male mice were challenged with a single dose of DOX prior to cardiac assessment 1 week or 4 weeks following DOX challenge. Adenoviruses encoding TBC1D15 or containing shRNA targeting Tbc1d15 were used for Tbc1d15 overexpression or knockdown in isolated primary mouse cardiomyocytes. Our results revealed that DOX evoked upregulation of TBC1D15 with compromised myocardial function and overt mortality, the effects of which were ameliorated and accentuated by Tbc1d15 deletion and Tbc1d15 overexpression, respectively. DOX overtly evoked apoptotic cell death, the effect of which was alleviated and exacerbated by Tbc1d15 knockout and overexpression, respectively. Meanwhile, DOX provoked mitochondrial membrane potential collapse, oxidative stress and DNA damage, the effects of which were mitigated and exacerbated by Tbc1d15 knockdown and overexpression, respectively. Further scrutiny revealed that TBC1D15 fostered cytosolic accumulation of the cardinal DDR element DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Liquid chromatography-tandem mass spectrometry and co-immunoprecipitation denoted an interaction between TBC1D15 and DNA-PKcs at the segment 594-624 of TBC1D15. Moreover, overexpression of TBC1D15 mutant (∆594-624, deletion of segment 594-624) failed to elicit accentuation of DOX-induced cytosolic retention of DNA-PKcs, DNA damage and cardiomyocyte apoptosis by TBC1D15 wild type. However, Tbc1d15 deletion ameliorated DOX-induced cardiomyocyte contractile anomalies, apoptosis, mitochondrial anomalies, DNA damage and cytosolic DNA-PKcs accumulation, which were canceled off by DNA-PKcs inhibition or ATM activation. Taken together, our findings denoted a pivotal role for TBC1D15 in DOX-induced DNA damage, mitochondrial injury, and apoptosis possibly through binding with DNA-PKcs and thus gate-keeping its cytosolic retention, a route to accentuation of cardiac contractile dysfunction in DOX-induced cardiotoxicity.

3.
Frontiers of Medicine ; (4): 1219-1235, 2023.
Article in English | WPRIM | ID: wpr-1010811

ABSTRACT

Heart failure with preserved ejection fraction (HFpEF) displays normal or near-normal left ventricular ejection fraction, diastolic dysfunction, cardiac hypertrophy, and poor exercise capacity. Berberine, an isoquinoline alkaloid, possesses cardiovascular benefits. Adult male mice were assigned to chow or high-fat diet with L-NAME ("two-hit" model) for 15 weeks. Diastolic function was assessed using echocardiography and noninvasive Doppler technique. Myocardial morphology, mitochondrial ultrastructure, and cardiomyocyte mechanical properties were evaluated. Proteomics analysis, autophagic flux, and intracellular Ca2+ were also assessed in chow and HFpEF mice. The results show exercise intolerance and cardiac diastolic dysfunction in "two-hit"-induced HFpEF model, in which unfavorable geometric changes such as increased cell size, interstitial fibrosis, and mitochondrial swelling occurred in the myocardium. Diastolic dysfunction was indicated by the elevated E value, mitral E/A ratio, and E/e' ratio, decreased e' value and maximal velocity of re-lengthening (-dL/dt), and prolonged re-lengthening in HFpEF mice. The effects of these processes were alleviated by berberine. Moreover, berberine ameliorated autophagic flux, alleviated Drp1 mitochondrial localization, mitochondrial Ca2+ overload and fragmentation, and promoted intracellular Ca2+ reuptake into sarcoplasmic reticulum by regulating phospholamban and SERCA2a. Finally, berberine alleviated diastolic dysfunction in "two-hit" diet-induced HFpEF model possibly because of the promotion of autophagic flux, inhibition of mitochondrial fragmentation, and cytosolic Ca2+ overload.


Subject(s)
Male , Mice , Animals , Heart Failure/drug therapy , Stroke Volume/physiology , Ventricular Function, Left/physiology , Berberine/therapeutic use , Disease Models, Animal , Mitochondrial Dynamics , Myocardium , Homeostasis
4.
Article in Chinese | WPRIM | ID: wpr-994572

ABSTRACT

Objective:To explore the team construction and treatment strategy of the Diabetic Foot-Multidisciplinary Team.Methods:The clinical data of 19 patients with severe ischemic diabetic foot treated by our Diabetic Foot-Multidisciplinary Team Center from Apr 2021 to Mar 2022 were collected, and the overall amputation rate, above-ankle major amputation rate, minor amputation rate and mortality, Diabetic Foot-Multidisciplinary Team consultation discipline participation rate and treatment participation degree were retrospectively analyzed.Results:Nineteen patients (15 males and 4 females) were enrolled, aged 26 to 94 (68.6±14.2). All were with severe ischemic diabetic foot ulcer:Rutherford grade 5 or up and dysfunction in 2 or more organs. Complications included arteriosclerosis obliterans of the lower extremities in 18 cases, heart diseases in 18, hypertension in 15, and renal insufficiencies in 10. The overall amputation rate was 36.8%, major amputation rate in 21.1%, minor amputation rate in 15.8%, and mortality rate was 15.8%. A total of 16 disciplines participated in Diabetic Foot-Multidisciplinary Team; the main participating disciplines were vascular surgery (19 times), endocrinology (12 times), and cardiology (11 times). The main treatment disciplines were vascular surgery (14 times), plastic surgery (3 times), and cardiology (2 times).Conclusion:For the diagnosis and treatment of diabetic foot, it is necessary to set up a multidisciplinary team as early as possible to control the causes of diabetic foot ulcer, prevent the recurrence of diabetic foot ulcer, reduce the mortality and amputation rate, and improve the quality of life of patients.

5.
Chinese Journal of Oncology ; (12): 338-345, 2019.
Article in Chinese | WPRIM | ID: wpr-805230

ABSTRACT

Objective@#To detect the effect and regulatory mechanism of human ether à go-go related gene 1 (Herg 1) knockdown on the proliferation and invasion of osteosarcoma (OS).@*Methods@#We constructed a recombinant adenovirus vector (Ad5-Herg1-shRNA) expressing short hair RNA (shRNA) against Herg1 and tested the knockdown efficiency. Then, the effects of Herg 1 knockdown on the proliferation, growth and invasion of osteosarcoma were measured by using cell counting kit-8 (CCK-8), wound healing assay, Transwell assay and xenograft model of nude mice, respectively. Tandem affinity purification, mass spectrometry and dual luciferase reporter assay were used to find out the molecules interacted with Herg1. Western blot was used to detect the expressions of large tumor suppressor gene (LATS1), p-LATS1, Yes-associated protein (YAP) and p-YAP in cells after infection of Ad5-Herg1-shRNA.@*Results@#Compared to Ad5-control-shRNA, Ad5-Herg1-shRNA dramatically inhibited the expression of Herg1 in OS cells. The result of CCK8 array demonstrated that 143B cell vitalities of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (65.47±3.90)% and (79.90±1.52)%, significantly lower than (100.00±6.14)% of Ad5-control-shRNA group. Meanwhile, U2OS cell vitality of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (69.69±1.36)% and (76.72±2.75)%, significantly lower than (100.00±3.01)% of Ad5-control-shRNA group (all P<0.001). The results of wound healing array showed that 143B cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (33.03±2.88)% and (36.47±4.16)%, significantly lower than (97.78±2.28)% of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (68.07±0.90)% and (73.97±1.25)%, significantly lower than (96.50±1.12)% of Ad5-control-shRNA group (all P<0.001). The results of Transwell showed that 143B cell invasion numbers of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 36.50±12.15 and 44.83±7.62, significantly lower than 195.33±19.68 of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 21.83±7.99 and 22.85±7.08, significantly lower than 83.33±12.36 of Ad5-control-shRNA group (all P<0.001). The results of xenograft model of OS showed that the tumor volume and weight of Ad5-Herg1-shRNA group were significantly smaller than of Ad5-control-shRNA group after 14 days and 5 weeks of inoculation, respectively (P<0.001). Moreover, knockdown of Herg1 inhibited the metastasis of OS cells. In mechanism, Herg1 protein interacted with NF2 protein. Knockdown of Herg1 significantly suppressed the expression levels of LATS1 and YAP protein, and promoted the phosphorylation of LATS1 and YAP in OS cells (all P<0.001).@*Conclusion@#Our findings suggest that Herg1 participates in the proliferation and motility of OS cells and may serve as a potential therapeutic target for osteosarcoma patients.

6.
Article in Chinese | WPRIM | ID: wpr-608797

ABSTRACT

Objective To study the infection rate of fusobacterium nucleatum cancer re appeared in patients with colorectal cancer before and after radiotherapy,and the changes after cancer recarrence.Methods A total of 20 persons receiving physical examination were recruited in the control group and collected the stool specimens,and 40 colorectal cancer patients were selected in the study group.All of the subjects in the study group were collected stool specimens before operation 3 days and after operation 5 day,after radiation therapy 7 days and 30 days.The patients were followed-up 1 year.The bacterial fluid was collected by filtration,and real-time fluorescence quantitative PCR was used to detect the expression of fusobacterium nucleatum gene in feces.Results The positive rate of fecal fusobacterium fusiformis was 30% in the study group and 5% in the control group.The gene relative expression of 12 colorectal cancer patients before operation 3 days and after operation 5 days,after radiation therapy 7 days and 30 days were 5.20±0.34,8.50±0.45,1.20±0.22,0.20±0.15.The fusobacterium nucleatum gene expression of 12 patients with positive fusobacterium after operation 5 days was significantly increased compared with that before operation 3 days(t=10.419,P=0.001),which after radiation therapy 7 days and 30 days was significant lower than that before operation 3 days(t=12.728,P=0.001;t=25.889,P=0.001).Six patients recurred among 1 year,the fusobacterium nucleatum gene expression was 7.2±0.56,which was significant higher than that after radiation therapy 7 days.Conclusion The infection of fusobacterium nucleatum might be a risk factor for colorectal cancer,and the gene relative expression might be an early warning indicator of recurrence.

7.
Article in Chinese | WPRIM | ID: wpr-482022

ABSTRACT

OBJECTIVE To investigate the arsenic trioxide(As2O3)-induced oxidative damage to mitochondrial DNA (mtDNA) in mouse oocytes and possible mechanisms. METHODS ① For in vitro assay,the mouse oocytes were denuded from ovaries of normal mice and incubated in medium for 20 h in different treatment groups:control,As2O3 1 and 2 μmol · L- 1,N-acetylcysteine (NAC) 5 mmol · L-1, 2,2,6,6-tetramethyl-1-piperidinyloxy(Tempo)1 mmol · L-1, As2O3(1 and 2 μmol · L-1)+NAC 5 mmol · L-1,As2O3 (1 and 2 μmol · L-1)+Tempo 1 mmol · L-1. ② For in vivo assay,mice were subjected to ip injection with physiological saline (normal control),As2O3 1 and 2 mg · kg-1,or As2O3 (1 and 2 mg · kg-1)+NAC 200 mg · kg-1, respectively. After 60 d,all the mice were sacrificed and their ovaries were quickly excised. Intracellular reactive oxygen species(ROS) levels were determined by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The oxidative damage to mtDNA was induced using enzyme-linked immunosorbent assay(ELISA)for 8-hydroxy-2′-deoxyguanosine(8-OHdG). The expression of DNA polymerase γ(Polγ)and mitochondrial transcription factor A(mtTFA)was detected by Western blotting and the vitality of lysosomes was monitored by β-galactosidase(β-Gal)Assay Kit. RESULTS ①In vitro experiments,As2O3 elevated 8-OHdG levels of mtDNA in mouse oocytes accom? panied by increased levels of ROS (P<0.05),but co-treatment with NAC or Tempo significantly reduced ROS and 8- OHdG levels (P<0.05). Meanwhile, the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05),but were markedly elevated by the addition of NAC or Tempo (P<0.05). ②In vivo assay,As2O3 elevated ROS as well as 8-OHdG levels of mtDNA in mouse oocytes,while the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05). Co-treatment with NAC significantly reduced ROS and 8-OHdG levels,but markedly elevated Pol γ and mtTFA levels(P<0.05). Besides,a notable increase in β-Gal activity was shown in As2O3-treated mouse oocytes in vitro (P<0.05),while antioxidants efficiently reduced the activity (P<0.05). However,no significant changes were observed in the in vivo study. CONCLUSION The oxidative damage to mtDNA induced by As2O3 in mouse oocytes may be mediated by ROS and associated with down-regulation of protein levels of Pol γ and mtTFA as well as increment of lysosomal activity.

8.
Practical Oncology Journal ; (6): 368-371, 2015.
Article in Chinese | WPRIM | ID: wpr-499315

ABSTRACT

Tumor derived microparticles are released by activated or apoptotic tumor cells.They are ex-tracellular vesicles which are 0.1~1.0μm in diameters.Tumor derived microparticles carry abundant bioactive molecules,such as nucleic acids and proteins,which resemble that of the parental cell.In this review,we summa-rize the role of tumor derived microparticles in the occurrence,development,diagnosis and treatment of tumor.

9.
Article in Chinese | WPRIM | ID: wpr-426248

ABSTRACT

Objective To summarize the nursing methods and effect of artificial femoral head replacement in treatment of osteoporotic interttochanteric fracture in elderly patients. Methods 55 patients with esteoporotic intertrochanteric fracture were selected in our hospital from February 2007 to February 2011.All patients were cared by targeted preoperative and postoperative nursing,helping them conducting functional training in time,giving discharge counseling and follow-up.The hip functional recovery of patients was evaluated by using the Harris evaluation standard,and the excellent rate of surgery was also evaluated. Results After the targeted care,55 patients went through the perioperative period safely,the average length of stay was ( 14.0 ± 3.1 )days,a total of 5 cases (9.09%)patiants suffered varying degrees of complications which were improved after symptomatic treatment.Patients were followed up for 3 to 36 months,only 2 patients died of cerebral vascular accident,all patients gained good recovery of hip function.Harris score was (84.45 ± 9.38),the excellent rate of surgery reached 78.18%. Conclusions Atificial femoral head replacement proves significant effect in treatment of osteoporotic intertrochanteric fractures in elderly patients,but with the accompanying surgical compliuations,special care and timely functional exercise methods should be paid attention to.

10.
Article in Chinese | WPRIM | ID: wpr-423286

ABSTRACT

Epidemiology,as a major subject in the field of public health science,plays a pivotal role in the construction and development of disease prevention and control system.It is also vital for the public health system to improve the emergency response ability and to cultivate high-quality talents.After analyzing current situation of graduate education of epidemiology,we found some problems.In our research,deepgoing dissection was carried out and possible solution was provided.

11.
Article in Chinese | WPRIM | ID: wpr-393530

ABSTRACT

Objective To study the relationship between the oncosis of pancreatic acinar cells and activa-tion of macrophage in rat model of acute pancreatitis (AP). Methods The pancreatic acinar cells were isolated by two-step enzyme digestion, and then they were divided into control group, AP group and test group. Pancreatic acinar cells were cultured with caerulein in AP group, with caerulein and endothelin in test group, and with culture medium in control group. The oncesis rate of the pancreatic acinar cells was detected after acridine orange and ethidium bromide fluorescent staining. The supernatant was collected to detect the release of amylase and lactate dehydrogenase (LDH). The macrophages were cultured with 1 ml of supematant for 6 hours, and then the protein level of tumor necrosis factor-α (TNF-α) was measured by ELISA. Results Few oncotic pancreatic acinar cells were observed in the control group, and the levels of amylase and LDH secreted by pancreatic acinar cells and TNF-α secreted by macrophage were (1175±165)kU/L, (846±118)U/L and (36±5)μg/L, respectively. Oncotic pancreatic acinar cells were observed in AP group, and the levels of amylase, LDH and TNF-α were (7130±680) kU/L, (4262±626) U/L and (155±18) μg/L, respectively, which were significantly higher than those in control group (t = 5.184, 4.277, 3.665, P < 0.05). The levels of amylase, LDH and TNF-α were even higher in test group, and they were (9240±1177) kU/L, (6937±893)U/L and (268±35)μg/L, respectively, which were significantly higher than those in AP group (t = 2.251, 2.825, 2.843, P < 0.05). Conclusions The release of amylase was changed as the oncosis of pancreatic acinar cells occurred. The secretion of TNF-α was along with the degree of oncosis of pancreatic acinar cells. The results of the study indicate that a relationship exists among the inflammatory response of macrophage, the release of contents of pancreatic acinar cells and the oncosis of the pancreatic acinar cells.

12.
Article in Chinese | WPRIM | ID: wpr-382066

ABSTRACT

Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.

13.
Article in Chinese | WPRIM | ID: wpr-554128

ABSTRACT

Objective:To set up an effective and simple purification method to obtain highly purified prokaryotic protein of PDCD5 and study its stability. Methods:Recombinant PDCD5 protein expressed in E. coli was accumulated as an inclusion body. After washing, the inclusion body was denatured, renatured, digested with thrombine and then purified by two steps of chromatography. The purity of the products was analyzed by capillary electrophoresis and the stability was identified by SDS-PAGE. Results:Capillary electrophoresis showed that the purity of protein was 100%, and molecular weight was 15 800 with pI 5.9. Further bioactivity assay indicated that the purified PDCD5 could enhance the apoptosis of HL 60 cells withdrawing cytokine, which was in a dose dependent manner. Stability analysis showed that the PDCD5 protein was sensitive to temperature and easy to degrade at 4 ℃ and 25 ℃. However, it was relatively stable at -20 ℃ or lyophilized. Conclusion:Highly purified and stable recombinant PDCD5 protein was obtained, which lays a foundation for the functional study and application investigation of PDCD5 .

14.
Article in Chinese | WPRIM | ID: wpr-560576

ABSTRACT

Objective: To develop a reporter gene system based on transient transfections with a NF-?B responsive reporter gene to detect the bioactivity of IL-1? and IL-1 receptor antagonist.Methods: NF-?B reporter and Dual-Luciferase assays were applied to measure the bioactivity of IL-1? and IL-1 receptor antagonist in mouse EL4 cells(some subclones of EL4 cells expressed high level of IL-1 receptor on cell surface).pNF-?B-luc and pRL-TK, used as an internal control,were co-transfected into EL4 cells and then the IL-1? was added.Results: The results indicated that IL-1? was able to induce the expression of this luciferase,which could be blocked by IL-1 receptor antagonist. The optimal dose of IL-1? was 5(?g/L) in Dual-Luciferase assay,whose bioactivity can be effectively inhibited by IL-1ra at 50 ?g/L.Conclusion: We have established a new method to detect the bioactivity of IL-1? and IL-1 receptor antagonist,which can give repeatable results.

15.
Chinese Journal of Hematology ; (12): 301-303, 2002.
Article in English | WPRIM | ID: wpr-261406

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effects of a novel human chemokine-like factor 1 (CKLF1) on stem cell/progenitor cells.</p><p><b>METHODS</b>Human bone marrow mononuclear cells were separated by Ficoll (1.077 g/ml), and cultured in suspension and semisolid colony culture. The effects of CKLF1 on CFU-GM and CFU-Mix were observed.</p><p><b>RESULTS</b>The number of CFU-GM was significantly increased in 10 groups by addition of CKLF1 plasmid supernatant. The mean value was 234.81 +/- 98.71/1 x 10(5) cells, 2.42 fold of control group (P < 0.05). The mean value of CFU-Mix in groups of negative control, CKLF1, PHA, GM-CSF and G-CSF were 82.00 +/- 20.25, 105.76 +/- 36.70, 146.63 +/- 27.09, 143.33 +/- 60.23 per 1 x 10(5) cells, respectively, no statistical differences were seen between control and CKLF1 groups. The CD(34)(+) cells were detected by FACS. The average percentage in the groups of normal control, CKLF1, PHA and GM-CSF were (0.75 +/- 0.62)%, (1.32 +/- 0.87)%, (0.15 +/- 0.02)%, and (0.29 +/- 0.23)%, respectively. Compared with control, no significant differences were seen between each group (P > 0.05).</p><p><b>CONCLUSION</b>Novel chemokine-like factor 1 can increase the proliferation of CFU-GM, which indicated that CKLF1 could increase the proliferation of human bone marrow hematopoietic progenitor cells and colony formation.</p>


Subject(s)
Adult , Humans , Cells, Cultured , Chemokines , Pharmacology , Hematopoietic Stem Cells , Cell Biology , MARVEL Domain-Containing Proteins
16.
Article in Chinese | WPRIM | ID: wpr-355110

ABSTRACT

<p><b>BACKGROUND</b>To investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.</p><p><b>METHODS</b>The recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.</p><p><b>RESULTS</b>The recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.</p><p><b>CONCLUSIONS</b>IL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.</p>


Subject(s)
Animals , Chick Embryo , Female , Humans , Mice , Rabbits , Adjuvants, Immunologic , Cells, Cultured , Genetic Vectors , Immunoglobulin A , Immunoglobulin G , Interleukin-6 , Genetics , Allergy and Immunology , Lung , Allergy and Immunology , Mice, Inbred BALB C , Recombination, Genetic , Vaccinia virus , Allergy and Immunology , Metabolism , Physiology , Virus Replication
17.
Article in Chinese | WPRIM | ID: wpr-411053

ABSTRACT

Objective: To study the expression and localization of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis. Methods: Using monoclonal antibody against TFAR19, the expression level and cell localization of TFAR19 were examined by fluorescence microscope, confocal laser scan microscope(CLSM) and flow cytometry. Simultaneously, we also analyzed the relationship of TFAR19 protein with phosphatidylserine (PS) externalization and cell nuclear DNA fragmentation. Results: The level of TFAR19 proteins expressed in TF-1 cells treated with GM-CSF withdrawal was significantly increased compared with normal TF-1 cells, then translocated rapidly from cytoplasm to the nucleus of cells. Appearance of TFAR19 in the nucleus of apoptotic cells preceded the detection of PS externalization and DNA fragmentation. Conclusion: Nuclear translocation of TFAR19 protein is one of the earliest events of cell apoptotic process. These data provided a new clue to further approach to the biological function of TFAR19 and study of cell apoptosis.

18.
Article in Chinese | WPRIM | ID: wpr-411035

ABSTRACT

Objective: in order to provide rapid and reliable method. Methods: Encoded Annexin Ⅴ cDNA was amplifyed from U937 cDNA libary by PCR and then subcloned into E coli expression vector. MS2-Annexin Ⅴ fusion protein could be overexpressed in E coli. The MS2 bacteria protein could be removed by thrombin digestion.The mature Annexin Ⅴ was obtained by ion exchange chromatography and the FITC labled Annexin Ⅴ could be used in the detection of apoptosis. Results:Up to 37% of the total bacterial proteins was rhAnnexin Ⅴ as showed by SDS-PAGE. The purification of Annexin Ⅴ is over 99%. The FITC labled Annexin Ⅴ could efficiently detect apoptosis. Conclusion: We successfully established the technique procedure of obtaining a large quantity of Annexin Ⅴ and provided the basic routine for popularizing the detection of apoptosis' with high effciency.

19.
Article in Chinese | WPRIM | ID: wpr-544273

ABSTRACT

Objective:To analyze a novel antigenic determinant of Homo sapiens CMTM1-v17 protein and prepare polyclonal antibody.Methods:By means of DNA recombination method, the C-terminal(The coding sequences of 118-149 residues) of CMTM1-v17 gene was inserted into pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli XL-1 Blue to express the fusion protein of GST/CMTM1-v17. The GST tag of the purified fusion protein was removed by enzymatic cleavage with thrombin. The polyclonal anti-CMTM1-v17 antibody were prepared by immunizing rabbits with the purified protein. Highly-specific, polyclonal anti-CMTM1-v17 antibodies was purified by affinity chromatographic column matrix coupled with antigen. The specificity of the anti-CMTM1-v17 antibody was identified by Western blot and immunohistochemistry.Results:The polyclonal antibody showed specific response to CMTM1-v17 by Western blot analysis and immunohistochemistry. These results suggested that the polyclonal antibody can be used for further studies of CMTM1-v17 function.Conclusion:A polyclonal anti-CMTM1-v17 antibody has been synthesized successfully.

20.
Article in Chinese | WPRIM | ID: wpr-581694

ABSTRACT

In order to improve the expression efficiency of recombinant IL~6 in eukaryotic cells, we constructed a new vector pcDIIL-6 to express IL-6 in eukaryotic cells on the basis of an eukaryotic vector pcDNA3IL-6 by introducin g the first intron of human?-globin gene into the downstream of CMV promoter in the pcDNASIL - 6. The IL-6 level expressed by pcDIIL - 6 is 7. 5 times higher than that of pcDNA3IL - 6. On the other hand , we studied the influence of pAdVAntage vector co - transfection on the expression level of IL-6 in eukaryotic cells and found the level of IL-6 is 3. 6 times higher than that of pcDNA3IL - 6. The results we got are beneficial to express cytokines with high efficiency in eukaryotic cells.

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