Acupuncture Points ; Diagnosis, Differential ; Humans ; Medicine, Chinese Traditional

Objective:To express human interferon-?2b gene and to explore the feasibility of expressing human gene in plant cells.Methods:The hIFN-?2b coding sequence was amplified by PCR with specific primers and plasmid pBV889 was used as a template,subcloned into middle vector pMD18-T and binary vector pBI121 to obtain plant expression vector pBIFN. The pBIFN was transformed into Agrobacterium tumefaciens strain LBA4404. Then hIFN-?2b gene was introduced into Ginseng callus cells via Agrobacterium-mediated transformation. The positive cells were screened by G418. The transgenic Ginseng calli were confirmed by PCR,RT-PCR,Western blot and WISH/VSV system.Results:Stable integration of the hIFN-?2b gene in the Ginseng callus cells′ genome was confirmed by PCR analysis. RT-PCR analysis showed that there were transcription products. Western blot implied that the given protein was hIFN-?2b. WISH/VSV system assay showed that the expressed hIFN-?2b possessed relatively lower bioactivity.Conclusion:HIFN-?2b has been expressed in transgenic Ginseng calli, which facilitates further investigation of improving the curative effect of orally administered hIFN-?2b.

Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250－700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.

OBJECTIVETo study the blood enriching effects of Paeoniae Radix Rubra and Paeoniae Radix Alba, paeoniflorin and albiflorin on mouse model of blood deficiency caused by γ-ray radiation.

METHODBuild mouse model of blood deficiency induced by γ-ray radiation. Paeoniae Radix Rubra and Paeoniae Radix Alba were given during modeling. The amount of WBC was detected af- ter the treatment. Based on the result of WBC and paeoniflorin content, albiflorin content in Paeoniae Radix Rubra and Paeoniae Radix Alba, the same model and the same method were used to comparatively study the effect of blood enriching of paeoniflorin and albiflorin.

RESULTOn the 7th day, the amount of WBC in model mice treated with 2 g x kg(-1) Paeoniae Radix Alba and 2 g x kg(-1) Paeoniae Radix Rubra significantly increased compared with that of model group (P < 0.05). In another experiment with the same model, the amount of WBC in model mice treated with 120 mg x kg(-1) paeoflorin and 120 mg x kg(-1) albiflorin significantly increased (P < 0.05) compared with that of model group on the 7th day. On the 10th day, the amount of WBC in rats treated with 120 mg x kg(-1) paeoflorin increased significantly (P < 0.05) compared with that of model group. Compared with the same dose of paeoniflorin, the amount of WBC in mice treated with albiflorin had no significant difference.

CONCLUSIONAll Paeoniae Radix Alba, Paeoniae Radix Rubra, paeoniflorin and al- biflorin can raise the amount of WBC and have the effect of enriching blood induced by radiation, while paeoniflorin and albiflorin have a similar result in this model. The result indicated that both paeoniflorin and albiflorin are effective constituents in Paeoniae Radix Alba, and paeoniflorin work as the common effective constituent in both Paeoniae Radix Rubra and Paeoniae Radix Alba.

Animals ; Bridged-Ring Compounds ; pharmacology ; Gamma Rays ; adverse effects ; Glucosides ; pharmacology ; Leukocyte Count ; Leukocytes ; cytology ; drug effects ; radiation effects ; Male ; Mice ; Monoterpenes ; pharmacology ; Rats

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; Glycosylation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo evaluate antisense technology for human telomerase inhibition in the treatment of endometrial cancer.

METHODSAn antisense oligodeoxynucleotides (AODN) directed against the human telomerase transcriptase (hTERT), designed and synthesized to serve as a telomerase inhibitor, was transfected into endometrial carcinoma cell line HEC-1A by lipofectin. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to test the expression of hTERT mRNA and hTERT protein before and after transfection. Telomerase activity was tested by telomeric repeat amplification protocol. The proliferation and growth of HEC-1A were also studied by methyl thiazolyl tetrazolium and cell growth curve before and after transfection.

RESULTSAODN could down-regulate the expression of hTERT mRNA and protein, inhibiting telomerase activity and proliferation of endometrial cancer cell line in a dose- and period-dependent manner.

CONCLUSIONAntisense oligodeoxynucleotides of human telomerase transcriptase definitely inhibits the proliferation of endometrial cancer cell line. Telomerase inhibitor may thus become a new gene therapeutic agent for endometrial carcinoma.

Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Endometrial Neoplasms ; pathology ; therapy ; Female ; Genetic Therapy ; Humans ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; analysis ; Telomerase ; antagonists & inhibitors ; genetics

OBJECTIVETo observe the clinical efficacy and safety on moderate and severe persistent allergic rhinitis treated with acupuncture.

METHODSSixty-six patients of moderate and severe persistent allergic rhinitis were randomized into an acupuncture group (34 cases) and a western medication group (32 cases). In the acupuncture, group, acupuncture was applied to Dazhui (GV 14), Feishu (BL 13), Pishu (BL 20), Ganshu (BL 18) and Shenshu (BL 23) in the prone, retained for 20 min; then in the supine, at Baihui (GV 20), Yintang (GV 29), yingxiang (LI20) Taichong (LR 3) and Hegu (LI 4), retained for 20 min. Acupuncture was given once every two days, three times a week, continuously for 8 weeks. In the western medication group, cetirizine hydrochloride was taken orally, 10 mg each time, once every day, continuously for 8 weeks. Separately, before treatment, after the treatment of 1 and 2 months and in 1 month after treatment, the total nasal symptom score (TNSS), the scores in the emotion rating scale for Ganzangxiang of TCM (ERSG) and the rhinoconjunctivitis quality of life questionnaire (RQLQ) were observed in the patients of the two groups. The clinical efficacy was compared between the two groups.

RESULTS(1) For TNSS, the results after 1 and 2 months treatment and in 1 month after treatment were all, reduced as compare with that before treatment separately in the two groups (P < 0.05, P < 0.01) The result after 2 months treatment was lower than that after 1 month treatment in the acupuncture group (P < 0.05). In 1 month after treatment, the result in the acupuncture group was lower than that in the western medication group (P < 0.05). (2) For ERSG, the score after 2 months treatment was lower than that before treatment in the two groups (both P < 0.05). The score after 2 months treatment in the acupuncture group was lower than that in the western medication group (P < 0.05). (3) For RQLQ, the score after 1 month treatment was lower than that before treatment and the score after 2 months treatment was lower than that after 1 month treatment in the two groups (all P < 0.05). The score after 1 and 2 months treatment and in 1 month after treatment in the acupuncture group was lower than that in the western medication group separately (all P < 0.05). (4) The total effective rate was 91.2% (31/34) in the acupuncture group and was 90.6% (29/32) in the western medication group, without significant difference between the two groups (P > 0.05).

CONCLUSIONAcupuncture is the safe and effective intervention on moderate and severe persistent allergic rhinitis. Compared with the western medicine group, the efficacy in the acupuncture group presents much more advantageous at its durability.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Female ; Humans ; Male ; Middle Aged ; Quality of Life ; Rhinitis, Allergic ; diagnosis ; therapy ; Young Adult

OBJECTIVETo observe the improvements in constipation differentiated as intestine system syndrome and lung system syndrome treated with electroacupuncture (EA) and explore their correlation.

METHODSSeventy cases of severe functional constipation were randomized into an EA group and a pseudo-EA group, 35 cases in each one. In the EA group, the needles were inserted deeply at Tianshu (ST 25) and Fujie (SP 14) on bilateral sides, and directly went to parietal peritoneum. EA was attached to the needles, dense-disperse wave, 2 Hz/15 Hz, and 0.1 to 1.0 mA. The perpendicular insertion was done at Shangjuxu (ST 37), 25 mm in depth. After qi arrival, the needle was lifted, thrusted and rotated once every 10 min, for 3 times totally. The needles were retained for 30 min. In the pseudo-EA group, the pseudo-points lateral to Tianshu (ST 25), Fujie (SP 14) and Shangjuxu (ST 37) on bilateral sides were punctured shallowly. The electric stimulation was pretended to connect but with the electric wire cutting off. The needles were retained for 30 min. The treatment was given 5 times weekly in the first two weeks and 3 times weekly in the later 6 weeks. Totally, 28 treatments were required. TCM intestine and lung syndrome scale was used for evaluation. The changes in TCM syndromes were observed before and after treatment in the patients of the two groups.

RESULTSThe total score of intestine and lung syndrome and the score of individual syndrome were all reduced after treatment as compared with those before treatment in the two groups (both P<0.01). The improvements of the EA group in the total score of intestine system syndrome, the scores of large. intestine syndrome and stomach syndrome, the total score of lung system syndrome and the score of lung dysfunction in dispersing and descending syndrome were superior to those of the pseudo-EA group (P<0.01, P<0.05). The differences in the scores of lung qi deficiency syndrome and throat syndrome were not significant between the two groups (all P>0.05). Simultaneously, the very strong positive correlation (P<0.01) and positive linear correlation (P<0.01) were presented in the total score of intestine and lung syndrome of the two groups.

CONCLUSIONEA obviously improves in intestine system syndrome (including large intestine syndrome and stomach syndrome), as well as lung system syndrome (lung dysfunction in dispersing and descending) in the treatment of constipation. But the improvements are not apparent in the treatment of lung qi deficiency and throat disorder. Additionally, the obvious correlation is displayed in the improvements in intestine and lung syndrome.

Acupuncture Points ; Adult ; Aged ; Constipation ; physiopathology ; therapy ; Electroacupuncture ; Female ; Humans ; Intestines ; physiopathology ; Lung ; physiopathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo evaluate the effect of integrative medical treatment (IMT) with serial Chinese recipes on quality of life (QOF) of rehabilitation stage in SARS patients.

METHODSEighty-five SARS patients of rehabilitation stage were enrolled in the clinical study. They were divided into the IMT group (62 patients received serial Chinese recipes and western medicine) and the control group (23 patients received western medicine alone). The serial Chinese recipes were given according to patients' syndrome, one dose per day for oral intake for 3 weeks. QOF scoring in patients was observed.

RESULTSQOF scoring in the IMT group before treatment was not significantly different from that in the control group. After 3 weeks treatment, it improved to some extent in both groups, but the improvement in the IMT group was superior to that in the control group in respect of total score and score of psychologic emotional factors.

CONCLUSIONSerial Chinese recipe could improve QOF of rehabilitation stage in SARS patients.

Adolescent ; Adult ; Aged ; Convalescence ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Ofloxacin ; therapeutic use ; Phytotherapy ; Quality of Life ; Ribavirin ; therapeutic use ; Severe Acute Respiratory Syndrome ; drug therapy ; immunology ; rehabilitation

Objective To investigate the relationship between killer cell immunoglobulin-like receptors(KIR)and HLA by distribution of KIR gene in leukemia patients and their siblings who have same HLA-A/B/DR typing.Methods KIR genotypes were detected by PCR-SSP on 78 patients and their siblings who have same HLA-A/B/DR typing.Results There were 48.72%in 78 patients who had same KIR genotypes with their siblings while the 44.87%patients had different KIR genotypes with their siblings.There was no difference in frequency between patients and their siblings(P＞0.05).There were no differences in frequency among chronic myelocytic leukemia(CML),non acute lymphoblagtic leukemia(NALL)and acute lymphoblagtie leukemia(ALL)but the frequency of KIR2DS4 in CML was higher than others.Condusion The KIR gene and HLAⅠ antigen are heredity independently and relatively stable.The factor of disease has little effect on KIR gene.