OBJECTIVE:To determine plasma concentration of irbesartan by HPLC with fluorescence detection.METHO-DS:Samples were separated on Diamonsil C18 with mobile phase consisted of 0.02mol?L-1 KH2PO4-acetonitrile(50∶50) at a flow rate of 1.0mL?min-1.The fluorescence detector was set at an excitation wavelength of 250nm and an emission waveleng-th of 375nm.The column temperature was 40℃.RESULTS:The linear range for irbesartan was from 1.0 to 1000ng?mL-1 (r=0.999 8).The mean relative recoveries of irbesartan at low,middle and high concentrations(2.0,50.0,and 500.00ng? mL-1) were 97.4%,106.1% and 101.6%,respectively.Both intra-day and inter-day RSD were below 10%.CONCLUSION:The method is simple,rapid,accurate and specific,and it can be used for the plasma concentration determination and pharmacokinetic study of irbesartan.

Protein kinase D (PKD) is a novel family of serine/threonine kinases and diacylglycerol (DAG) receptors and has been documented in a variety of cellular processes. To get high purity catalytic domain of PKD1 (PKD1-cat) for crystallography study, the GST-tagged PKD1-cat gene was cloned into a baculovirus transfer vector pFastBac1 (donor plasmid). When the recombinant plasmid was transformed into DH10Bac competent Escherichia coli, which contains a baculovirus shuttle vector (bacmid), transposition occurs to generate a recombinant bacmid containing the gene of interest (GST-PKD1-cat). The recombinant bacmid DNA was transfected into Spodoptera frugiperda Sf9 insect cells to generate recombinant baculovirus, which was then amplified through multiple rounds of infection in Sf9 cells. After that, Trichoplusia ni insect cells in suspension culture were infected with baculoviral stock at a multiplicity of infection (MOI) of 5 PFU/cell. SDS-PAGE and Western blotting analysis confirmed the detection of a 68 kDa protein by the glutathione S-transferase (GST) monoclonal antibody. The recombination protein was purified by Glutathione sepharose affinity chromatography and cleaved by PreScission Protease to remove GST tag, and a highly pure 42 kDa protein which was consistent with the molecular weight of the expected PKD1-cat protein was detected on SDS-PAGE. The activity of purified PKD1-cat protein was determined by in vitro PKD kinase assay. Our data showed that the kinase activity increased with the concentration of purified PKD1-cat protein. These results showed that the truncated recombinant PKD1-cat protein was highly active and pure, and could potentially be used for solving 3D structure of this protein by Nuclear Magnetic Resonance (NMR) or crystallography.
Animals ; Baculoviridae ; Blotting, Western ; Catalytic Domain ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; Plasmids ; Protein Kinase C ; chemistry ; Recombinant Proteins ; chemistry ; Spodoptera

Objective To introduce a proposal that can standardize the clinical information of urine manifestation in Chinese medicine.Methods A hypothesis of symptomatic units was proposed in this paper,herein,the attribute,name and definition of the symptomatic units of urine manifestations in the clinical records of Chinese medicine in the past dynasties were summarized according to the stipulation of logic classification and denomination of a scientific term.Results The 23 symptomatic units,2 bounds and their assembly can express 3867 descriptions on the clinical manifestation of urine.Conclusion The symptomatic unit is a feasible hypothesis that can simplify the clinical information of Chinese medicine.The attribute extraction,denomination and definition may become one of the effective procedures to standardize the clinical information of Chinese medicine.

Objective　To evaluate the treatment of in-stent restenosis with rotational atherectomy and balloon angioplasty. Methods　The rotational atherectomy and 4～6 atm low pressure balloon angioplasty was performed in 3 patients with in-stent restenosis and follow up after treatment. Results　All cases were succeeded. The bradycardia occurred in one patient was quickly disappeared without treatment, two other patients were found no effect on heart rate, hemodynamic performance, global LV function, or regional wall motion. No complications, angina, death or other coronary event occurred during the follow up for 6～12 months. Two of them was performed coronary angiography after 6 months and showed the diameter of target vessel was less than 30% as compared with that on coronary angiography which performed immedately after operation. Conclusion　The management of in-stent restenosis in target vessels using a combination of rotational atherectomy and balloon angioplasty is safe and efficient.

Objective To explore clinical effects of laparoscopy applied in the female patients with infertility. Methods 468 cases of female infertility patients were diagnosed and treated by laparoscopy and 168 cases received follow-up survey. Results Among those infertility patients, the patients of normal pelvic cavity and the abnormal one were 49(10.47% ) and 419( 89.53% ). Among those patients of abnormal pelvic cavity .fallopian tube disease in 276 cases(65.87% ) was at the first place. The constituent ratio of endometriosis,ovary tumor,polycystic ovary,uterine myoma,female genital tract malformation and pelvic tuberculosis was 14. 80% ,5. 97% ,4. 30% ,3. 82% , 3. 34% and 1.91% .respectively. The patients of primary infertility and the secondary ones were 208 and 260 cases. In secondary infertility patients 69.23% (180/260) of patients were caused due to fallopian tube diseases,and the constituent ratio was higher than that in primary infertility 46.15% (96/208) (x2 = 14.05 ,P <0.05),while in primary infertility,the constituent ratio of endometriosis and normal pelvic cavity were 20.19% ,15.38% ,which were higher than that in secondary infertility respectively(x2 = 11. 78,11. 16, all P < 0.05). The pregnancy rate of 168 cases followed-up was 45.24% (76/168).47.37% ,84.21% pregnancy occurred within 6,12 months with laparoscopy. The shortest and longest time of pregnancy was 2 and 30 months, respectively. The median was 8months. Conclusion Laparoscopy playd an important role in diagnosis of infertility and improvement of pregnancy rate.

0.05) while other indicators did not. All the data were simply related to each other. The primary influencing factors to NDS score 1, in decreasing order of impact were volume of infarction, position of infarction, hs-CRP and FBS. While the primary influencing factors to NDS score 3 at the end of 1 month treatment, in decreasing order of impact, were NDS score 1, size of infarction, FG, hs-CRP and TC. Conclusions Volume of infarction and hs-CRP are very important determining factors in both NDS score 1 at the beginning of the treatment and NDS score 3 at the end of 1 month treatment. However, in determining NDS score 3 at the end of 1 month treatment, the impact of FG is greater than hs-CRP. The other influencing factors include FBS, TC and so on. Therefore we conclude that it is helpful to recover from neurologic deficit through lowing these influencing factors directly by medication.

Objective Investigate the postmortem stability of the six immunohistochemical markers of fibronectin(Fn),fibrinogen(Fg),C5 complement(C5), myoglobin(Mb), actin(HHF35)and desmin(Dm)for the postmortem diagnosis of myocardial infarction.Method The areas of depletion ofMb、HHF35 and Dm,and the positive reaction areas of Fn、Fg and C5 in the ischemic myocardium were studied with immunohistochemistry,image analysis technique and statistical system.The postmortem stability of the six immunohistochemical markers was compared.Results The specimens of normal myocardium kept at 4℃ for 1 to 2 days showed homogenous brown reactions for Dm, HHF35 and Mb; depletion of Dm, HHF35 and Mb became evident when these specimens were kept at 4℃ for 3 days postmortem, and the depletion area increased with the lapse of postmortem interval; the depletion area of Dm, HHF35 and Mb in ischemic myocardial tissues also increased with the lapse of postmortem interval;the positive reaction areas of Fg, C5 and Fn in ischemic myocardial tissues decreased with the lapse of postmortem interval.Fg became negative when the ischemic myocardium were kept at 4℃ for 2 weeks postmortem,C5 became negative when kept at 4℃ for 3 weeks postmortem,but Fn remained positive when kept at 4℃ for 4 weeks postmortem.No positive reactions for Fg,C5 and Fn could be found in normal myocardium when kept at 4℃ for different time intervals. The image analysis result showed that the positive reaction areas decreased with the lapse of postmortem interval. Conclusion The Dm and HHF35, Mb showed least postmortem stability, easily influenced by autolysis, only suitable for detection in fresh corpses(1to 2dayspostmortem) ;Fgislittlebitbetter,suitableforcorpsesat4℃ 1weekpostmortem ;C5isbet ter,suitableforcorpsesat 4℃ 2weekspostmortem ;Fnisthebestmarker ,suitableforcorpsesat 4℃ 4 weekspostmortem .

BACKGROUND: The expression of main histocompatibility antigen in cornea of rats is similar to that of human. It is verified in early experiments that penetrating corneal transplantation in rats is more repetition and reliability, and the operation difficulty is lower than that in mice.OBJECTIVE: To establish rat model of penetrating corneal transplantation and analyze the reasons of complications during model preparation and the corresponding managements and probe into the method for improving the success rate of rat model of corneal transplantation.DESIGN: Randomized controlled experiment was designed.SETTING: Department of Ophthalmology , Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was performed in Department of Ophthalmology in Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from March to May 2004. Seventysix outbred female Wistar rats of two months old were employed and randomized into experimental group and control group with 38 rats in each,without eye disease, of clean grade, body mass varied from 180 to 200 g and their right eyes were taken as acceptors. Thirty-eight SD female rats were employed and their both eyes were taken as donors. All of the animals were provided from Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology.METHODS: Central penetrating corneal transplantation in situ was adopted and the model was established on the right eye of Wistar rat. Ten minutes before operation, tropicamide eye drops was administrated twice to amplify adequately the pupil of the right eye up to 4.0 mm. According to body mass, general anesthesia was done with abdominal injection of 3.0 mL/kg chloral hydrateand dicaine eye drops was applied twice 5 minutes before the operation with 0.1 volume fraction. Routine sterilization was done on the head of rat, the eyes kept in horizontal level and respiration kept smooth. The clip in dental-ophthalmology department was used to fix the posterior of the operated eye and make it to be semiluxated. The ring driller 3.5 mm in diameter was used to collect corneas of both eyes in SD rat as the implant tissue and place in culture dish with endothelial part upward, covered with methyl cellulose of 20 g/L mass weight concentration. The ring driller, 3.0 mm in diameter was used to collect the cornea of the right eye in Wistar rat to be the graft foreman, and the implant tissue was sutured into the graft foreman on the right eye of Wistar rat with 10-0nylon thread intermittently. Eight stitches were done in corneal transplantation in the experimental group and 6 stitches in the control group. After operation, ofloxacin eye drops was administrated and the operated eye was covered with plaster. Corneal rejection was observed and the evaluation was done in 3 items, named, turbidity, edema and neogenetic vessel. The total score of 3 items was taken as the rejection index on the day. Rejection was determined if the rejection index on the day ≥5 or corneal turbidity reached the 3nd grade. x2 and Fisher definite probability methods were used for statistical examination.plant tissue and corneal rejection.RESULTS: Of 76 Wistar rat acceptors, 5 rats were dropped out, among which, 1 rat was died accidentally after operation in the experimental group, 2 rats were died accidentally after operation in the control and 2 rats were injured cornea on the operated eyes due to fighting. But by supcomplications after corneal transplantation: In the experimental group, the number with anterior adhesion of sclera, non-round pupil; limited wound healing and non-formation of anterior chamber was lower than that in the implant tissue and corneal rejection: the survival time of corneal implant tissue was similar basically in both experimental group and the control [(8.9±2.3), (8.6±2.3) days, P ＞ 0.05]. Rejection presented in both groups in 16 days after corneal transplantation and the incidence of rejection was 100%.CONCLUSION: With stitches maintained and without intervention, the acute rejection is similar in rat corneal transplantation and human corneal transplantation. It is explained in the experiment that 8 stitches in corneal transplantation in which the rat implant tissue of 3.5 mm matches graft foreman of 3.0 mm can reduce the complications of rat corneal transplantation and becomes a satisfactory animal model of corneal transplantation.Skillful microscopic operation techniques, satisfactory operation instruments and adequate amplilfied pupil can reduce to the most extent the postoperation complications in rats.

Objective To Analyze CD4+ cell count which embodies curative effect in HIV and patients with AIDS, who have treated with TCM for half a year. Methods According to CD4+cell count, the patients were divided into 4 phases. Their CD4+cell count were analyzed before and after the treatment. Results 1.Rank sun test showed that CD4+cell count were significantly improved in people who used TCM treatment. On the whole, CD4+cell count was(317.76±175.61) in 1 cu mm before treatment, which was(350.60±175.92) in 1 cu mm after treatment, P<0.01. 2. Ridit showed that patients whose CD4+cell count more than 500 were in phase I. Their R=0.614, and the 95%confidence interval was 0.5702 to 0.6579. Patients whose CD4+cell count more than 350 and less than 500 were in phase II. Their R=0.575, and the 95%confidence interval was 0.5439 to 0.6062. Patients whose CD4+cell count more than 200 and less than 350 were inphase III. Their R=0.460 and the 95%confidence interval was 0.4347 to 0.4849. Patients whose CD4+cell count less than 200 were in phase IV. Their R=0.428, and the 95%confidence interval was 0.3971 to 0.4589. There was no overlap between phase III, phase IV and phase I, phase II in 95% confidence interval. Conclusion TCM has the advantages in strengthening vital qi, but that is worse than western medicine in effort of expelling pathogen.