AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimag...AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.展开更多
Objective： The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells. Methods： Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal an...Objective： The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells. Methods： Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal antibody was prepared by hybridoma cell technique. Recombinant adenovirus Ad.yrdC carrying yrdC gene was constructed by using the AdEasy adenoviral vector system. Recombinant adenovirus Ad.yrdCshRNA mediated yrdCshRNA was prepared by RNA interference technology. Gastric adenocarcinoma BGC-823 cells of moderate differentiation were transfected and absorbance of the transfected cells was calculated at 490 nm by methyl thiazolyl tetrazolium （MTT） method. Results： A value of the transfected Ad.yrdC group was significantly greater than that of the non-transfected and transfected Ad.Null groups, and A value of Ad.yrdCshRNA group was significantly lower than that of the non-transfected and transfected Ad.Null groups. Conclusion： Expression of yrdC gene has a function of promoting the proliferation of gastric carcinoma cells.展开更多
The cDNA expression libraries derived from a highly metastatic cell subline Anip-973 and from its parental cell line, low metastatic AGZY-83a were screened by monoclonal antibodies (MoAbs) seperately against these two...The cDNA expression libraries derived from a highly metastatic cell subline Anip-973 and from its parental cell line, low metastatic AGZY-83a were screened by monoclonal antibodies (MoAbs) seperately against these two cell lines. A positive clone(H4-D) from the Anip-973 cDNA library was isolated and its nucleotide sequence was determined. This clone contained 978 bp with an open reading frame of 318 bp encoding a polypeptide consisting of 106 amino acids. The H4-D cDNA sequence showed 85% homology with a human propionyl-CoA carboxylase α-chain. In Western bloting analysis, the MoAb H4 recognized 2 bands(15 KDa and 27 KDa) of Anip-973 cell membrane Protein. The mRNA expression of H4-D was higher in AniP-973 cells than that in AGZY-83a cells. The metastatic Potential of Anip-973 cells was markedly decreased after being pretreated with MoAb H4. The above findings indicated that H4-D has a certain relationship with the metastatic phenotype of AniP-973 cells.展开更多
AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in ...AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.展开更多
AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the D...AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.展开更多
A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signa...A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.展开更多
IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method ...IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method from 3H11 hybridoma cells, using 5′ primers for leader sequences. The 3H11 VL gene was then inserted into humanmouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.RESULTS DNA sequence analysis indicated that the cloned genes included the whole leader sequences and the mature Ig variable region encoding sequences. After gene transfection, transient expression of chimeric light chain protein was detected.CONCLUSION DNA sequences and transient expression indicated that the cloned gene was functional. This work laid basis for constructing 3H11 humanmouse chimeric antibody in the future..展开更多
基金National Natural Science Foundation of China,No.39700175
文摘AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.
基金Supported by a grant from the General Program of National Natural Science Foundation of China （No, 30371403）.
文摘Objective： The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells. Methods： Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal antibody was prepared by hybridoma cell technique. Recombinant adenovirus Ad.yrdC carrying yrdC gene was constructed by using the AdEasy adenoviral vector system. Recombinant adenovirus Ad.yrdCshRNA mediated yrdCshRNA was prepared by RNA interference technology. Gastric adenocarcinoma BGC-823 cells of moderate differentiation were transfected and absorbance of the transfected cells was calculated at 490 nm by methyl thiazolyl tetrazolium （MTT） method. Results： A value of the transfected Ad.yrdC group was significantly greater than that of the non-transfected and transfected Ad.Null groups, and A value of Ad.yrdCshRNA group was significantly lower than that of the non-transfected and transfected Ad.Null groups. Conclusion： Expression of yrdC gene has a function of promoting the proliferation of gastric carcinoma cells.
文摘The cDNA expression libraries derived from a highly metastatic cell subline Anip-973 and from its parental cell line, low metastatic AGZY-83a were screened by monoclonal antibodies (MoAbs) seperately against these two cell lines. A positive clone(H4-D) from the Anip-973 cDNA library was isolated and its nucleotide sequence was determined. This clone contained 978 bp with an open reading frame of 318 bp encoding a polypeptide consisting of 106 amino acids. The H4-D cDNA sequence showed 85% homology with a human propionyl-CoA carboxylase α-chain. In Western bloting analysis, the MoAb H4 recognized 2 bands(15 KDa and 27 KDa) of Anip-973 cell membrane Protein. The mRNA expression of H4-D was higher in AniP-973 cells than that in AGZY-83a cells. The metastatic Potential of Anip-973 cells was markedly decreased after being pretreated with MoAb H4. The above findings indicated that H4-D has a certain relationship with the metastatic phenotype of AniP-973 cells.
基金Supported by the National 863 Project of China,No.102-09-01-02National Natural Science Foundation of China,No.39770827
文摘AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.
文摘AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.
文摘A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.
文摘IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method from 3H11 hybridoma cells, using 5′ primers for leader sequences. The 3H11 VL gene was then inserted into humanmouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.RESULTS DNA sequence analysis indicated that the cloned genes included the whole leader sequences and the mature Ig variable region encoding sequences. After gene transfection, transient expression of chimeric light chain protein was detected.CONCLUSION DNA sequences and transient expression indicated that the cloned gene was functional. This work laid basis for constructing 3H11 humanmouse chimeric antibody in the future..
