Summary: The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats ...Summary: The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats were randomly assigned to two groups: control group and RA group. The rats in RA group was intraperitoneally injected with all trans-retinoic acid (500 μg/kg every day) for consecutive 3 days after birth, while those in the control group were not subjected to intervention. Immunohistochemical assay was performed to locate the expression of PDGF. mRNA levels of PDGF were measured by reverse transcription polymerase chain reaction (RT-PCR) at age of 1, 3, 5, 7, 10, 14, 21 days. The method of radial alveolar counts (RAC) was used to measure the amount of the alveoli of the lungs. It was found that with increasing days, levels of PDGF-A and PDGF-B changed to verying degrees. RA could elevate significantly the expression levels of PDGF-A mRNA and protein (P<0.01), but not affect the expression levels of PDGF-B mRNA and protein markedly (P>0.05). It is suggested that PDGF might play an important role in lung development. RA can stimulate lung development through increasing the expression levels of PDGF-A mRNA and protein.展开更多
In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-39...In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.展开更多
Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of...Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.展开更多
Background Prospective mortality studies in the United States revealed that the mortality was elevated in diabetics compared to normal individuals following chronic spinal cord injury （SCI）. Our study was conducted ...Background Prospective mortality studies in the United States revealed that the mortality was elevated in diabetics compared to normal individuals following chronic spinal cord injury （SCI）. Our study was conducted to investigate the levels of platelet-derived growth factor （PDGF） of astrocytes in SCI in streptozotocin （STZ）-induced diabetic rats. Methods Thirty male Sprague-Dawley （SD） rats were randomly divided into 3 groups： SCI group, diabetic SCI group, and sham operation control group. We employed STZ-induced diabetic SD rats and a weight-drop contusion SCI model. The rats were sacrificed on day 7 after the induction of SCI. Immunohistochemistry and Western blotting analysis were used to detect the PDGF expression level. Basso, Beattie and Bresnahan locomotor rating scale （BBB） was also used to evaluate the neurological recovery level of the rats. Results PDGF positive astrocyte numbers were significantly higher and PDGF staining was more intensive in astrocytes in the SCI group than in the diabetic SCI group （P〈0.05）. The diabetic SCI group showed a slower recovery of motor function with a lower BBB score 7 days after acute spinal injury. Conclusions PDGF is an important factor for the recovery of neurological function after acute spinal injury and hyperglycemia in diabetic rats could depress the expression of PDGF in injured spinal cord. This may help to explain the slower recovery and higher mortality in diabetics after SCI.展开更多
Objective:Platelet-rich plasma(PRP)releases growth factors upon activation,which in turn accelerates healing and regeneration of the target tissue.However,PRP composition may vary according to the patient’s demograph...Objective:Platelet-rich plasma(PRP)releases growth factors upon activation,which in turn accelerates healing and regeneration of the target tissue.However,PRP composition may vary according to the patient’s demographics,and wider applications of PRP warrant product standardization.The current study aimed to examine variables influencing the platelet-derived growth factor BB(PDGF-BB)concentration in PRP.Methods:This observational study was conducted in the Department of Pathology and Dentistry at Swami Rama Himalayan University,a tertiary care hospital in northern India from December 2016 to November 2017.PRP was prepared from 40mL of whole blood from 35 individuals(22 women,13 men).Platelet counts,platelet indices(platelet distribution width,mean platelet volume)and PDGF-BB levels were measured,and platelet yield,platelet dose,and growth factor dose in PRP were also calculated.All parameters were analyzed using Pearson’s correlation coefficient.The association between PDGF-BB and PRP platelet count was evaluated using logistic regression.This study was approved by the Ethics Committee of Swami Rama Himalayan University(SRHU/HIMS/ETHICS/2016/103)on September 7,2016.Results:The mean platelet count,PDGF-BB concentration,platelet yield,platelet dose,and growth factor dose in PRP were 1317×10^(9)/L,30±9.89ng/mL,71.62±28.34%,6.5±3.5×10^(9),and 159.62±52.39ng/mL,respectively.Linear regression analysis indicated that PRP platelet counts were a good predictor for PGDF-BB(P<0.05;adjusted R^(2)=0.96.PRP platelet count was significantly positively correlated with PDGF-BB concentration(r=0.74,P<0.001),platelet yield(r=0.80,P<0.001),platelet dose(r=1,P<0.001),and growth factor dose(r=0.74,P<0.001).Conclusions:PRP has wide clinical applications associated with its healing and regenerative properties,and both the quality and quantity of PRP thus need to be standardized as per the requirements.Evaluating variables affecting PRP will thus aid pathologists and clinical practitioners.展开更多
AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astr...AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.展开更多
文摘Summary: The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats were randomly assigned to two groups: control group and RA group. The rats in RA group was intraperitoneally injected with all trans-retinoic acid (500 μg/kg every day) for consecutive 3 days after birth, while those in the control group were not subjected to intervention. Immunohistochemical assay was performed to locate the expression of PDGF. mRNA levels of PDGF were measured by reverse transcription polymerase chain reaction (RT-PCR) at age of 1, 3, 5, 7, 10, 14, 21 days. The method of radial alveolar counts (RAC) was used to measure the amount of the alveoli of the lungs. It was found that with increasing days, levels of PDGF-A and PDGF-B changed to verying degrees. RA could elevate significantly the expression levels of PDGF-A mRNA and protein (P<0.01), but not affect the expression levels of PDGF-B mRNA and protein markedly (P>0.05). It is suggested that PDGF might play an important role in lung development. RA can stimulate lung development through increasing the expression levels of PDGF-A mRNA and protein.
基金Shanghai Medical Development grant No. ZD99001 and aGrant (SFB-542) from the Deutsche Forschungsgemeinschaft.
文摘In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30300151 ）.
文摘Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.
基金This work was supported by a grant from Zhejiang Provincial Natural Science Foundation （No. Y2080348）.
文摘Background Prospective mortality studies in the United States revealed that the mortality was elevated in diabetics compared to normal individuals following chronic spinal cord injury （SCI）. Our study was conducted to investigate the levels of platelet-derived growth factor （PDGF） of astrocytes in SCI in streptozotocin （STZ）-induced diabetic rats. Methods Thirty male Sprague-Dawley （SD） rats were randomly divided into 3 groups： SCI group, diabetic SCI group, and sham operation control group. We employed STZ-induced diabetic SD rats and a weight-drop contusion SCI model. The rats were sacrificed on day 7 after the induction of SCI. Immunohistochemistry and Western blotting analysis were used to detect the PDGF expression level. Basso, Beattie and Bresnahan locomotor rating scale （BBB） was also used to evaluate the neurological recovery level of the rats. Results PDGF positive astrocyte numbers were significantly higher and PDGF staining was more intensive in astrocytes in the SCI group than in the diabetic SCI group （P〈0.05）. The diabetic SCI group showed a slower recovery of motor function with a lower BBB score 7 days after acute spinal injury. Conclusions PDGF is an important factor for the recovery of neurological function after acute spinal injury and hyperglycemia in diabetic rats could depress the expression of PDGF in injured spinal cord. This may help to explain the slower recovery and higher mortality in diabetics after SCI.
基金This work was financially supported by Swami Rama Himalayan University。
文摘Objective:Platelet-rich plasma(PRP)releases growth factors upon activation,which in turn accelerates healing and regeneration of the target tissue.However,PRP composition may vary according to the patient’s demographics,and wider applications of PRP warrant product standardization.The current study aimed to examine variables influencing the platelet-derived growth factor BB(PDGF-BB)concentration in PRP.Methods:This observational study was conducted in the Department of Pathology and Dentistry at Swami Rama Himalayan University,a tertiary care hospital in northern India from December 2016 to November 2017.PRP was prepared from 40mL of whole blood from 35 individuals(22 women,13 men).Platelet counts,platelet indices(platelet distribution width,mean platelet volume)and PDGF-BB levels were measured,and platelet yield,platelet dose,and growth factor dose in PRP were also calculated.All parameters were analyzed using Pearson’s correlation coefficient.The association between PDGF-BB and PRP platelet count was evaluated using logistic regression.This study was approved by the Ethics Committee of Swami Rama Himalayan University(SRHU/HIMS/ETHICS/2016/103)on September 7,2016.Results:The mean platelet count,PDGF-BB concentration,platelet yield,platelet dose,and growth factor dose in PRP were 1317×10^(9)/L,30±9.89ng/mL,71.62±28.34%,6.5±3.5×10^(9),and 159.62±52.39ng/mL,respectively.Linear regression analysis indicated that PRP platelet counts were a good predictor for PGDF-BB(P<0.05;adjusted R^(2)=0.96.PRP platelet count was significantly positively correlated with PDGF-BB concentration(r=0.74,P<0.001),platelet yield(r=0.80,P<0.001),platelet dose(r=1,P<0.001),and growth factor dose(r=0.74,P<0.001).Conclusions:PRP has wide clinical applications associated with its healing and regenerative properties,and both the quality and quantity of PRP thus need to be standardized as per the requirements.Evaluating variables affecting PRP will thus aid pathologists and clinical practitioners.
基金Supported by the Biomedical Research Councilthe Institute of Bioengineering and Nanotechnology,the Republic of Singapore
文摘AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
