RESUMO
Currently,the research or publications related to the clinical comprehensive evaluation of Chinese patent medicine are increasing,which attracts the broad attention of all circles. According to the completed clinical evaluation report on Chinese patent medicine,there are still practical problems and technical difficulties such as unclear responsibility of the evaluation organization,unclear evaluation subject,miscellaneous evaluation objects,and incomplete and nonstandard evaluation process. In terms of evaluation standards and specifications,there are different types of specifications or guidelines with different emphases issued by different academic groups or relevant institutions. The professional guideline is required to guide the standardized and efficient clinical comprehensive evaluation of Chinese patent medicine and further improve the authority and quality of evaluation. In combination with the characteristics of Chinese patent medicine and the latest research achievement at home and abroad,the detailed specifications were formulated from six aspects including design,theme selection,content and index,outcome,application and appraisal,and quality control. The guideline was developed based on the guideline development requirements of China Assoication of Chinese medicine. After several rounds of expert consensus and public consultation,the current version of the guideline has been developed.
Assuntos
Medicina Tradicional Chinesa , Medicamentos sem Prescrição , Consenso , China , Padrões de Referência , Medicamentos de Ervas ChinesasRESUMO
<p><b>OBJECTIVE</b>To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells.</p><p><b>METHODS</b>The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants.</p><p><b>RESULTS</b>Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX.</p><p><b>CONCLUSIONS</b>CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.</p>
Assuntos
Animais , Cricetinae , Humanos , Antígenos CD , Genética , Alergia e Imunologia , Metabolismo , Antígeno CTLA-4 , Cárie Dentária , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia , Metabolismo , Vacinas de DNA , Genética , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To detect the immunoreactivity of targeted fusion anti-caries DNA vaccine pGJA-P in vitro, and the ability to enhance the immune responses compared with the non-targeted fusion anti-caries DNA vaccine pGLUA-P.</p><p><b>METHODS</b>The CHO cells were transfected with pGJA-P and the expression of recombinant protein in cultured supernatants were detected using Western blotting. 5 to 6-month-old female Japanese rabbits were immunized with either pGJA-P or pGLUA-P via either intramuscular injection (i.m.) or intranasal route (i.n.). The sera and saliva were collected and the antibody responses were checked by ELISA. The effect of immune sera on the synthesis of water-insoluble glucan by glucosyltransferase of S. mutans was examined.</p><p><b>RESULTS</b>The expressed protein could response to specific anti-GTF antibody. The antibody responses in serum generated by pGJA-P via i.m. were significantly higher than those generated by pGLUA-P (P < 0.01). The antibody responses in saliva generated by pGJA-P via i.n. were significantly higher than those generated by pGLUA-P (P < 0.01). The higher mucosal antibody response induced by pGJA-P via i.m. compared with pGLUA-P (P < 0.01) was detected. The immune sera of rabbits immunized by pGJA-P via i.m. most significantly inhibited the synthesis of water-insoluble glucan by glucosyltransferase.</p><p><b>CONCLUSIONS</b>The recombinant protein expressed by pGJA-P had the immunoreactivity to anti-GTF antibody. pGJA-P could induce faster and higher specific mucosal SIgA antibody responses via i.n. or serum IgG antibody responses via i.m. compared with non-targeted DNA vaccine, pGLUA-P. High titres of specific mucosal antibodies were found in rabbits immunized with pGJA-P via i.m. The immune sera of rabbits immunized by pGJA-P via i.m. displayed the ability of inhibiting the synthesis of water-insoluble glucan by glucosyltransferase.</p>
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Animais , Cricetinae , Feminino , Coelhos , Proteínas de Bactérias , Alergia e Imunologia , Células CHO , Cárie Dentária , Glucosiltransferases , Alergia e Imunologia , Metabolismo , Imunoglobulina G , Sangue , Glicoproteínas de Membrana , Alergia e Imunologia , Proteínas Recombinantes de Fusão , Alergia e Imunologia , Streptococcus mutans , Alergia e Imunologia , Transfecção , Vacinas de DNA , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in situ. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and pGLUA-P, a fusion anticaries DNA vaccine.</p><p><b>METHODS</b>pGJA-P was administrated intramuscularly or intranasally to rats, and the expression of recombinant protein was detected by immunohistochemistry technique. Wistar rats were fed a cariogenic diet and orally infected with S. mutans, then immunized with pGJA-P or pGLUA-P via the intramuscular or intranasal route. All rats received a booster immunization 2 weeks later. At the termination of the experiment, blood and saliva samples were collected for assay of antibodies by ELISA and jaws were obtained for caries evaluation by the Keyes method.</p><p><b>RESULTS</b>Recombinant protein could be detected in muscle in intramuscularly immunized rats and in nasal mucosa in intranasally immunized rats. Rats immunized intramuscularly with pGJA-P had significantly higher serum IgG levels than others (P < 0.01). Rats immunized intranasally or intramuscularly with pGJA-P had significantly higher salivary IgA levels than others (P < 0.01). Keyes scores of pGJA-P groups were significantly lower than those of pGLUA-P groups and pCI groups (P < 0.01).</p><p><b>CONCLUSIONS</b>pGJA-P could be correctly expressed in vivo. pGJA-P generated increased humoral immune response and anticaries efficacy compared with pGLUA-P.</p>
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Animais , Feminino , Ratos , Plasmídeos de Bacteriocinas , Alergia e Imunologia , Cárie Dentária , Ratos Wistar , Proteínas Recombinantes de Fusão , Alergia e Imunologia , Streptococcus mutans , Alergia e Imunologia , Vacinas de DNA , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To construct and detect the targeted anti-caries fusion DNA vaccine pGJA-P encoding the A-P fragment of pac, glu fragment of gtfB and extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) gene for the targeting antigen to APC.</p><p><b>METHODS</b>The extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) genes were amplified by RT-PCR from human lymphocytes, and the genes were cloned into pUC(m-T) vector respectively. After sequencing, Fc region of human Iggamma(1) gene was cloned to the downstream of CTLA4 gene fragment as the recombinant plasmid pGJ. The fusion gene was then inserted into the plasmid pGLUA-P to get the recombinant plasmid pGJA-P. The CHO cells were transfected with liposome and the expression of fusion protein in cultured supernatants were detected using Western blotting.</p><p><b>RESULTS</b>The plasmids pGJ and pGJA-P carried the CTLA4-Ig fusion gene and CTLA4-Ig fusion gene, A-P fragment of pac gene and glu fragment of gtfB gene respectively. The expressed protein could response to specific anti-PAc antibody.</p><p><b>CONCLUSION</b>The targeted fusion anti-caries DNA vaccine pGJA-P is constructed successfully and expressed in eukaryotic cells correctly.</p>