目的建立重组人干扰素β1a(Recombimant human interferon beta1a,rhIFNβ1a)生物学活性MTS/PMS检测方法。方法将MTS和PMS偶联作为染色液,建立IFNβ1a生物学活性检测方法,对细胞浓度、细胞病变时间、MTS工作浓度和染色时间进行优化,并...目的建立重组人干扰素β1a(Recombimant human interferon beta1a,rhIFNβ1a)生物学活性MTS/PMS检测方法。方法将MTS和PMS偶联作为染色液,建立IFNβ1a生物学活性检测方法,对细胞浓度、细胞病变时间、MTS工作浓度和染色时间进行优化,并绘制效应曲线。对建立的方法进行重复性、准确性验证,并与结晶紫染色法进行比较。结果优化后的MTS/PMS法的最佳反应条件为:细胞浓度1×105个/ml,细胞病变时间24～36 h,MTS工作浓度2 mg/ml,染色时间40 min;A570/630值与IFNβ1a保护Wish细胞S型效应曲线的相关系数(R2)均达0.99以上。不同检测板、相同加样位置的变异系数在6%～20%之间;相同检测板、不同加样位置的变异系数在13%～17%之间;检测IFNβ1a细胞收集液的回收率在86%～121%之间。该法检测rhIFNβ1a生物学活性效应曲线呈反"S"型,线型较好,R2值均在0.99以上,均比结晶紫染色法的R2值高,且比结晶紫染色法更稳定。结论已建立了rhIFNβ1a生物学活性MTS/PMS检测方法,适用于常规定量测定rhIFNβ1a的生物学活性。展开更多
In vitro chemosensitivity testing of short term primary glioma cultures derived from brain biopsies is still in the research phase and has not yet found a place in clinical use. The main reasons for this slow progress...In vitro chemosensitivity testing of short term primary glioma cultures derived from brain biopsies is still in the research phase and has not yet found a place in clinical use. The main reasons for this slow progression are the small amounts of tissue available and the lack of a suitably sensitive assay capable of use in the clinical setting. This study examines whether the MTS and ATP cell survival assays, which determine cytotoxicity via colorimetric and luminescence analysis respectively, could potentially fulfill this role. Primary glioma cultures were tested for chemosensitivity using the MTS and ATP assays and were found to be generally sensitive to cisplatin and paclitaxel but relatively resistant to carmustine and etoposide. For both assays, LD50 values lay in the range 2 - 130 μg/ml but in the vast majority of cases, those obtained by the ATP assay were markedly lower those obtained by the MTS assay. Moreover, at cell numbers less than 2000 in the cases of paclitaxel and carmustine and less than 4500 in the case of cisplatin, these drugs were generally indicated as ineffective against the glioma cultures tested by the MTS assay but effective against these cultures by the ATP assay. These data clearly demonstrate that the ATP assay is more sensitive when estimating small cell numbers generated by primary glioma cultures from brain biopsies and more reliably detects higher kill rates by anticancer drugs. This study also supports the feasibility of using the ATP assay for chemosensitivity testing in a clinical setting.展开更多
Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin...Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells （SUVEC）. The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein （EGFP） fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase （15% to 20% increase） and fewer were in S phase （18% decrease） compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.展开更多
Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarde...Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.展开更多
文摘In vitro chemosensitivity testing of short term primary glioma cultures derived from brain biopsies is still in the research phase and has not yet found a place in clinical use. The main reasons for this slow progression are the small amounts of tissue available and the lack of a suitably sensitive assay capable of use in the clinical setting. This study examines whether the MTS and ATP cell survival assays, which determine cytotoxicity via colorimetric and luminescence analysis respectively, could potentially fulfill this role. Primary glioma cultures were tested for chemosensitivity using the MTS and ATP assays and were found to be generally sensitive to cisplatin and paclitaxel but relatively resistant to carmustine and etoposide. For both assays, LD50 values lay in the range 2 - 130 μg/ml but in the vast majority of cases, those obtained by the ATP assay were markedly lower those obtained by the MTS assay. Moreover, at cell numbers less than 2000 in the cases of paclitaxel and carmustine and less than 4500 in the case of cisplatin, these drugs were generally indicated as ineffective against the glioma cultures tested by the MTS assay but effective against these cultures by the ATP assay. These data clearly demonstrate that the ATP assay is more sensitive when estimating small cell numbers generated by primary glioma cultures from brain biopsies and more reliably detects higher kill rates by anticancer drugs. This study also supports the feasibility of using the ATP assay for chemosensitivity testing in a clinical setting.
文摘Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells （SUVEC）. The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein （EGFP） fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase （15% to 20% increase） and fewer were in S phase （18% decrease） compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.
文摘Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.
