BACKGROUND: It has been confirmed that Chinese medicine astragalus mongholicus and radix notoginseng can improve symptoms of lower limb ischemia. It is supposed that this kind of therapy is associated with the promotion effect of astragalus mongholicus and radix notoginseng on differentiation of bone marrow mesenchymal stem cells (BMSCs) into vascular endothelial cells.OBJECTIVE: To identify that whether astragalus mongholicus and radix notoginseng are effective in promoting the proliferation and differentiation of BMSCs into vascular endothelial cells. DESIGN, TIME AND SETTING: The in vitro differentiation experiment was performed at the Key Laboratory of Education Ministry, Dong Zhimen Hospital Affiliated to Beijing University of Chinese Medicine from April to June 2005. MATERIALS: Bone marrow blood from 23 patients with lower limb ischemia was collected routinely at the Dong Zhimen Hospital Affiliated to Beijing University of Chinese Medicine. The density gradient centrifugation was used to separate bone marrow mononuclear cells, I.e. BMSCs. Astragalus mongholicus injection (equal'to 2 g/mL crude drug, Diaojiuhong, China), Xuesaitongfen injection (main component radix notoginseng, Zhenbaodao, China), CD34 antibody (BD, USA) and FACS Calibur flow cytometer (BD, USA) were used in this study. METHODS: BMSCs were incubated with 3 mL low, middle and high doses of astragalus mongholieus (containing 4, 12 and 36 g/L crude drug), and 3 mL low, middle and high doses of Xuesaitong suspension (50, 100 and 200 mg/L radix notoginseng). BMSCs only cultured without drugs served as a blank control group. Medium was changed every other day, for successively 3 weeks.MAIN OUTCOME MEASURES: Three weeks later, cell morphology was observed under an inverted phase contrast microscope. CD34+ cell percentage was detected by flow cytometry.RESULTS: Three weeks after BMSC culture, most cells were spindle-shaped, fascicular, with shape and characteristics of endothelial cells. Compared with the blank control group, the percentage of CD34+ cells were significandy increased in the low and middle doses of astragalus mongholicus group and in the middle and high doses of radix notoginseng group (P < 0.05-0.01). CONCLUSION: Astragalus mongholicus and radix notoginseng have the function of promoting proliferation and differentiation of BMSCs into vascular endothelial cells in a dose dependent effect.

This study was aimed to observe the clinical efficacy of Jie-Du Tong-Mai (JDTM) decoction (i.e., detoxi-fication unclogg artery soup) of arteriosclerosis obliterans. A total of 246 arteriosclerosis obliterans cases with the pattern of blood stasis were selected and randomly divided into two groups. In the control group of 120 cases, 0.1 g of aspirin and 20 mg of simvastatin tablets were orally administrated, once a day. In the treatment group of 126 cas-es, JDTM decoction was combined on the basis of oral administration of aspirin and simvastatin tablets. After 8 weeks of treatment, observations were made on improvement of clinical symptoms and changes of ABI, Hs-CRP, TcpO2. The results showed that compared with the control group, clinical symptoms of the treatment group were obvi-ously improved (P< 0.05). And the ABI, CRP, TcpO2 were also obviously increased (P< 0.01). It was concluded that JDTM decoction was effective in the treatment of arteriosclerosis obliterans with the pattern of blood stasis. It al-so indirectly proved the guiding meaning of traditional Chinese medicine that evil causing stasis.

This study was aimed to discuss the external treatment of diabetic foot ulcer with traditional Chinese medicine (TCM) Yin-Huang-San (YHS) and to observe its promoting healing effect. Analysis was made on AGEs correlated with inflammatory factors and growth factors. A total of 60 patients with damp-heat-toxin pattern standard diabetic foot ulcer patients were randomly divided into the treatment group and control group. External application of YHS was given in the treatment group. And external application of metronidazole and glucose injection was given in the control group. The dressing was changed once a day. Blood was drawn 1 d before the treatment, and 7 d, 14 d, 21 d, 28 d after the medication. Improvements of patients’ clinical symptoms were observed. Dynamic monitoring was given on changes of hs-CRP, TNF-α, IL-1, VEGF, EGF, bFGF, PDGF and AGEs. The results showed that the total efficiency of the treatment group was 96.7%; and the total efficiency of the control group was 83.3%. Compared with the control group, external treatment with YHS can obviously improve clinical symptoms of diabetic foot ulcer (P<0.01), reduce AGEs and inflammatory factor, and increase the number of growth factors. AGEs had significant positive correlation with inflammatory factor; it had significant negative correlation with growth factors. It was concluded that YHS was effective in the treatment of diabetic foot ulcer patients with damp-heat-toxin pattern.

Objective To establish a multiplex suspension array for simultaneously detecting five species of important arbovirus: dengue virus (including four serotypes), japanese B encephalitis virus, west nile virus, tick-borne encephalitis virus and yellow fever virus. Method All published genomic sequences of the above arboviruses were collected from GeneBank, and then, the genus universal primers and specific probes were designed according to the conservative and specific NS5 region of the genus Flavivirus. The amine-modified probes were covalently coupled to different carboxylated microspheres by carbodiimide methods. By combining general RT-PCR with virus-specific probe hybridization, a Luminex xMAP system-based suspension array, which could simultaneously differentiate five arboviruses and discriminate 1～4 serotypes of dengue virus, was successfully established. Results A cutoff value twice higher than that of the background fluorescence was judged as positive reaction. RT-PCR products of many arboviruses were used to evaluate the specificity and repeatability of the array, the results showed that the assay was highly specific and had good repeatability. Meanwhile, the coefficient of variability was less than 8% from inter- and intra- assay, implying that both the repeatability and stability of the array were good. Furthermore, several viruses which had been quantitated by plaque assay were used to determine the array’s sensitivity. The results showed that the array’s detection limits were about 1.4 PFU for JBEV and YFV, 14 PFU for WNV respectively. 55 clinical and cultured samples were detected by the established assay, 16 out of the 55 samples were confirmed to be positive. There was only one sample which was not detected contrasted with conventional RT-PCR. However, it had advantages in detecting complex samples rapidly. Conclusion A multiplex suspension array for simultaneously detecting and serotyping five species of important arboviruses has been successfully developed, and it may play an important role in diseases diagnosis and prevention as well as epidemiological studies.

Objective To examine the role of mitochondrial energy-conversion efficiency in regulating the initiation and execution of the apoptotic process in outer hair cells(OHCs)of cochlea following exposure to intense noise.Methods Seventeen adult chinchillas were used in present study.The animals were randomly assigned to one of the two groups,12 animals were exposed to noise and the remaining 5 animals without exposure to noise served as normal control.For all the animals,3-nitropropionic acid solution(3-NP,50mmol/L),an irreversible inhibitor of succinate dehydrogenase(SDH),was dropped onto the round window of the cochlea of the right ear using a micro-syringe to inhibit the mitochondrial energy production,to serve as the test ear.Artificial perilymph solution(AP)was dropped onto the round window of the cochlea of the left ear to serve as the control ear.16h after application of 3-NP and AP,the animals were exposed to 75 pairs of impulse noise at 155 dB pSPL.The cochleae were harvested 2h after the noise exposure.The cochlear basilar membrane was stained with propidium iodide(PI),a DNA intercalating fluorescent probe used to visualize the morphologic viability of hair cell nuclei.All the specimens were examined with a fluorescence microscope.Results In the 3-NP-treated cochlea,medium degree of nuclear condensation of OHCs appeared to be the dominant nuclear pathology,and only a few OHCs showed nuclear fragmentation in the damaged area of the cochlea.In contrast,the AP-treated control ear exhibited a large quantity of nuclear fragments,and a small quantity of medium degree of nuclear condensation.Conclusion The present study indicates that mitochondrial energy-conversion function plays an important role in regulating the apoptotic process.Disruption of the mitochondrial energy can not deter the apoptotic process from initiation,but can slow down the pace of apoptotic progression in outer hair cells of the cochlea following exposure to intense noise.

Objective Apoptosis and necrosis are two forms of hair cell death in cochlea following exposure to intense impulse noise.The present study was designed to examine the activity of caspase-3,caspase-8 and caspase-9 in the basilar membrane of cochlea,and investigate the cellular signal pathway associated with noise-induced hair cell death in cochlea.Methods Twenty-seven adult chinchillas were used in present study.The animals were randomly assigned into test group(no.15) and control group(no.12).The animals in test group were exposed to noise for the detection of the activity of caspase-3,caspase-8 and caspase-9,respectively,each with 5 animals.Animals in control group were not exposed to noise.Chinchillas were exposed to impulse noise at 155 dB peak sound pressure level(pSPL) for 75 times.Two hours after noise exposure,both cochleae were perfused with approximately 30?l of freshly prepared solution containing one of the three caspases(caspase-3,-8 and-9) respectively using a micro-syringe.One hour after perfusion,the animals were sacrificed and the cochleae were harvested.The Corti organs were double stained with propidium iodide(PI),and a DNA intercalating fluorescent probe was used to visualize the morphologic viability of hair cell nuclei.All the specimens were observed with a fluorescence microscope.Results The normal cochlea did not exhibit green fluorescence for any of the three caspases in Corti organ.Strong caspase-3,-8 and-9 green fluorescence appeared in the outer hair cells with condensed or fragmented nuclei,a sign of apoptosis,whereas no fluorescence was observed for any of the three caspases in the swollen outer hair cell nuclei,a sign of necrosis,in the cochleae exposed to intense impulse noise.Conclusion It is indicated that caspase-9 is involved in the intrinsic and caspase-8 involved in the extrinsic apoptotic pathway which occur simultaneously in apoptotic outer hair cells.No caspase activation occurs in necrotic outer hair cell in the cochlea following exposure to intense impulse noise.

Objective To compare the prevalence of apoptosis and necrosis, and investigate the primary death pathway of outer hair cells of rat cochlea following styrene exposure. Methods Fourteen adult Long Evans rats were used in the present study. The animals were randomly assigned into test group (n=8) and control group (n=6). Animals in test group were exposed to styrene by gavage at 400 mg/kg (2g styrene was mixed with 1ml olive oil). Treatment was performed once a day, 5 days per week for 3 weeks. Animals in control group were fed by gavage the same volume of olive oil on an identical time schedule used for the test group. The auditory brainstem response (ABR) thresholds of both ears elicited with clicks were measured before and at the end of the 3-week styrene or olive oil treatment. After hearing was re-assessed, animals were sacrificed and cochleae were quickly removed from the skull. Following fixation, whole specimens comprising the basilar membrane with Corti's organ were separated from the modiolus. Apoptotic, necrotic and missing outer hair cells (OHCs) were distinguished by combined assays of nuclear staining with propidium iodide (PI), TUNEL assay and filamentous actin(F-actin)staining with FITC-phalloidin. Each Corti's organ was thoroughly examined by fluorescence microscopy. The numbers of damaged OHCs (apoptotic, necrotic and missing OHCs) were documented. Results Neither threshold shift of ABR nor sign of hair cell (HC) damage was found in the cochlea of control animals. The animals of test group showed both physiological and pathological changes in the cochleae following the 3-week styrene treatment. ABR testing revealed an average of 15 dB of threshold shifts. F-actin staining exhibited the maximal level of OHCs damage in the middle portion of Corti's organ. The major damage occurred in the third row of OHCs, followed by the second and first rows of OHCs. Three types of morphological changes in damaged OHC nuclei were revealed by PI labeling: nuclear condensation, nuclear swelling and nuclear missing. Strong TUNEL green fluorescence appeared in the OHCs with condensed nuclei. Quantitative analysis showed that the average number of apoptotic OHCs was approximately three times greater than the number of necrotic OHCs (P=0.01). Conclusion It is indicated that apoptosis is the primary death pathway of OHCs leading to generation of the cochlear lesion following styrene exposure.

Objective To discuss the causes and therapies for the complications induced by endovenous laser treatment (EVLT) in patients with varicose great saphenous vein. Methods From August 2003 to February 2007,totally 342 patients (530 limbs) with varicose great saphenous veins were treated by high ligation combined with EVLT in our hospital. Operative compilations were found in 186 of the limbs.The data of the 186 limbs were analyzed retrospectively. Results The patients were followed up for 6 months,during which 23 limbs showed skin bruises and/or hematoma,65 develped phlebitis,and 20 had skin burns; all of these cases were cured. In addition,77 limbs were diagnosed as having saphenous nerve damage (abnormal skin sensation),57 of them were cured and 20 were improved after treatments. Deep vein thrombosis complicated with pulmonary embolism was found in one case,who was improved after therapies. Conclusions To prevent the complications of EVLT combined with high ligation of the saphenous vein,intraoperative adjustment of laser power according to the surgical sites,gentle operation,postoperative administration of anticoagulants,and early rehabilitation are recommended.

Objective To quantitatively analyze the occurrence of apoptotic and necrotic outer hair cells (OHCs) and to evaluate the changes of these parameters with time after noise exposure. Methods Chinchillas were exposed to a narrow band noise at either 104 or 108 dB SPL for 1 hour. The animals were executed and dissected at either 1, 4 and 30 days after the noise exposure and the cochleas were collected for detection of OHC death mode. The apoptotic and necrotic OHCs were distinguished by examining the OHC nuclear morphology and confirmed by staining for caspase 3 activity or TUNEL assay. ABR thresholds for click stimuli were used to monitor changes in auditory function. Results The number of apoptotic cells were significantly greater than those of necrotic cells shortly after the noise exposure at day 1 for the 104 dB group, day1 and day4 for 108 dB group ( P =0 01, P =0 03, P =0 01) and the difference between the number of apoptotic cells and necrotic cells became statistically insignificant at day 4 and day 30 for the 104 dB group and day 30 for 108 dB group ( P =0 67, P =0 29, P =0 52). By day 30, apoptotic and necrotic pathologies continued, although in small quantity in both 104 dB group and 108 dB group. Conclusions The results of the study indicated that the early expansion of cochlear lesion is contributed primarily by apoptosis, whereas the later stage of lesion expansion is likely contributed equally by apoptosis and necrosis. The death of OHCs not only takes place during a noise exposure, but also continues for at least 30 days after noise exposure

BACKGROUND: Decoy receptor 1 (DcR1), which acts as decoy receptors, is resistant to apoptosis induction by related apoptosis inducing ligand (TRAIL). OBJECTIVE: To investigate the expression of the DcR1 (TRAIL-R3) protein in human herniated and normal lumbar intervertebral discs (IVD). METHODS: The expression and distribution of DcR1 proteins were assessed by using immunohistochemistry in 20 herniated lumbar IVD and 8 normal lumbar IVD tissues samples following discoidectomy from January to September 2010. RESULTS AND CONCLUSION: Percentage of nucleus pulposus and anulus fibrous cells with DCR1 expression was obviously more in the herniated group than in normal IVD group. The current results indicate that the expression of DCR1 rises after herniation.