3.Expression and antitumor activity of fusion protein RGD-TRAIL in Pichia pastoris.
Acta Pharmaceutica Sinica 2015;50(5):552-559
To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing α(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.
Antineoplastic Agents
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pharmacology
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Apoptosis
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Blotting, Western
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Cell Line, Tumor
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Chromatography, Affinity
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli
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Humans
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Oligopeptides
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biosynthesis
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pharmacology
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Pichia
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metabolism
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Plasmids
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Recombinant Fusion Proteins
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biosynthesis
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pharmacology
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TNF-Related Apoptosis-Inducing Ligand
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biosynthesis
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pharmacology
4.Research progress of electrospuning technique in constructing tissue engineering tracheal scaffold
Shu PAN ; Fei SUN ; Hongcan SHI
International Journal of Biomedical Engineering 2013;36(6):357-361
One of the key contents in tissue engineering trachea replacement research is the scaffold selection.This review summarizes the latest original literatures and investigations about electrospun technique as well as recent progress.To discuss the advantages and disadvantages of natural,synthetic and combined electrospun scaffolds,the versatility in material choosing and production methods is the unique superiority.For specific experimental or clinical objects,the further research is to choose a suitable polymer,to improve surface modification techniques and to control the dimension and arrangement of the fibrous structure of electrospun fibers,which can offer versatility and tissue specificity,and therefore provide a bright prospect for tissue engineering trachea.
6.Interventional heated chemotherapy for moderate and advanced huge primary hepatocellular carcinoma
Journal of Regional Anatomy and Operative Surgery 2013;(6):611-612,614
Objective To explore the effects of the therapeutic effects of interventional heated chemotherapy( IHC) for moderate and advanced huge primary hepatocellular carcinoma and compare with transcatheter arterial chemoembolization( TACE) . Methods The 68 pa-tients with moderate and advanced huge primary hepatocellular carcinoma accepted the treatment of IHC were considered as the observation group,and 32 patients managed TACE were regarded as the control group. The therapeutic effects,living quality,secondary surgery rates,The reduced level of serum AFP,survival rate,and toxic side effects of 2 groups were contrasted. Results The effective rate of observation group was obviously higher than that of control group (79. 4% vs 56. 3%,P <0. 05),and secondary surgery rates was increased(70. 6% vs 43. 8%,P<0. 01). The ratio of increased Karnofsky score was more than 20 in observation group,which was obviously higher than that of control group(35. 3% vs 12. 5%,P<0. 01). T The reduced level of serum AFP was more than 50% in observation group,which was higher than that of control group (52. 9% vs 25. 0%,P<0. 01). The survival rate of 12,18,24 months after treatment in control group was in-creased than that in control group individually (P<0. 01). Conclusion IHC is effective for huge primary hepatocellular carcinoma and in-creases the secondary surgery rates,which is one of the safe and effective methods for huge primary hepatocellular carcinoma.
7.Effects of high-fat-diet on the level of hormones related to glucose and lipid metabolism in rat plasma.
Xiang LI ; Reng-Fei SHI ; Shu-Jie LOU
Chinese Journal of Applied Physiology 2014;30(4):293-295
Animals
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Diet, High-Fat
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Glucagon
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blood
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Glucose
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metabolism
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Hormones
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blood
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Insulin
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blood
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Leptin
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blood
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Lipid Metabolism
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Male
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Rats
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Rats, Sprague-Dawley
8.Value of different biological markers in prediction of response to neoadjuvant chemotherapy in breast cancer
Fei XIE ; Bo ZHOU ; Yingming CAO ; Shu WANG ; Deqi YANG
China Oncology 2000;0(06):-
Background and purpose:Neoadjuvant chemotherapy is an excellent model for evaluation of predictive parameters.The goal of predictive parameters is to select patients who would most likely benefit from neoadjuvant chemotherapy.The objective of this study was to investigate the predictive value of biological markers for the patients' response to neoadjuvant anthracycline combined with taxanes chemotherapy.Methods:We investigated 160 patients with breast cancer who underwent 4 cycles of neoadjuvant anthracycline combined with taxanes chemotherapy,retrospectively.The expression of estrogen receptors(ER),progesterone receptors(PgR),Her2,Topo-Ⅱ and Ki-67 proteins were detected by immunohistochemical assay in core-needle biopsy specimens.The associations between biological markers as well as clinical and pathologic responses were analyzed.Results:The overall clinical response was 85%,including 28.8%(46/160) with clinical complete response(cCR) and 56.3%(90/160) with clinical partial response.The pathological complete response(pCR) was 14.4%.In the univariate analysis,absence of ER,PgR expression and over-expression of Her-2 were predictive of the cCR and pCR(P
9.Serum levels of interleukin-36 and its receptor antagonist in patients with psoriasis and their correlations with disease severity
Dan SHU ; Hongzhong JIN ; Baoxi WANG ; Fei SU ; Feng LI
Chinese Journal of Dermatology 2014;47(7):469-472
Objective To evaluate the relationship of serum levels of interleukin (IL)-36α,IL-36β and IL-36γas well as their receptor antagonist IL-36Ra with disease severity in patients with psoriasis.Methods Venous blood samples were collected from 45 patients with generalized pustular psoriasis (GPP),34 patients with psoriasis vulgaris (PV),and 37 healthy human controls.Enzyme-linked immunosorbent assay (ELISA) was performed to determine the serum levels of IL-36α,IL-36β,IL-36γ and IL-36Ra.Nonparametric Mann-Whitney test was conducted to compare the levels of IL-36 and IL-36Ra among these groups,and Spearman's rank correlation analysis to assess the relationship between the serum levels of IL-36 and IL-36Ra and disease severity.Results No statistical difference was observed in the serum levels of IL-36 or IL-36Ra among the patients with GPP,patients with PV,and healthy human controls.The serum levels of IL-36β and IL-36γ (given as the median ± interquartile range) were significantly higher in 27 patients with GPP during episodes of pustules ((12.101 ± 11.315) ng/L and (34.541 ± 15.580) ng/L respectively) and in 7 patients with severe GPP ((11.218 ± 9.318) ng/L and (38.536 ± 17.332) ng/L respectively) than in the healthy human controls ((5.355 ± 9.020) ng/L and (23.052 ± 22.410) ng/L respectively,P < 0.05 or 0.01).The serum level of IL-36γwas positively correlated with that of IL-36β in patients with GPP,patients with PV,and the healthy human controls (r =0.85,0.86,0.91,respectively,all P < 0.01),and both IL-36β and IL-36γserum levels were lowly and positively correlated with the severity of GPP (r =0.33,0.41,respectively,both P < 0.05).A positive correlation was also observed between the serum level of IL-36β and psoriasis area and severity index (PASI) scores in patients with PV (r =0.54,P < 0.01).Conclusions The serum levels of IL-363 and IL-36γ are lowly and positively correlated with disease severity in patients with GPP,suggesting that IL-36β and IL-36γplay an important role in the pathogenesis of GPP.
10.Construction of Recombinant Mice Factor Ⅶ-pPIC9K Vector and Expression in Pichia
Zhengrong YANG ; Fei HE ; Meng WANG ; Yongping SHU ; Tianming CHENG
Chinese Journal of Cancer Biotherapy 1995;0(02):-
Objective:To construct the yeast expressive vector of rmFⅦ, in which mFⅦ was mutated to inhibit coagulation without affecting the affinity for TF, and express it in Pichia pastoris. Methods:The full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method, site-direct mutated and restriction enzyme digested as design. Cloning into pPIC9K, electroporation of Gs115, in vivo screen of multiple inserts by G418 resistance, BMGY/BMMY are used for induction and expression of rmFⅦ in pichia pastoris. These proteins were also screened for functional activity. Results: Three different rmFⅦ-pPIC9K yeast expression vectors and it's aim protein were obtained,two kinds of proteins were found to be functional active as design. Conclusion:rmFⅦ protein can be expressed in pichia pastoris and it might facilitate the development of tumor-target molecule, and novel anti-agiogenesis drug study.