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Gu H.-S.,Shenzhen Second Hospital | Cheng Z.-A.,Guangzhou University of Chinese Medicine | Li Z.-Y.,Guangzhou University of Chinese Medicine | Zhou W.-Y.,Shenzhen Second Hospital | Zou X.-Y.,Shenzhen Traditional Chinese Medicine Hospital
Journal of Clinical Rehabilitative Tissue Engineering Research

BACKGROUND: The construction of adenovirus vector using Adeasy system is tedious with poor results due to the multiple restriction enzyme digestion, linkage, and screening. OBJECTIVE: To construct a recombinant adenovirus vector containing LMP-1 gene using Gateway technology. METHODS: Total RNA was extracted from rat osteoblasts and the LMP-1 gene was acquired by RT-PCR, the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology, then the entry clone and the expression vector were used to create the expression clone through the LR recombination reaction; at last the adenovirus expression clone was linearized by PacI and transfected to the 293A cell line. The target gene was compared to LMP-1 in the Genebank (Gene number: AF095585), and the expression of pAd-LMP-1 was observed. RESULTS AND CONCLUSION: The electrophoresis showed that the extracted RNA was integrated, and the purity was accorded with the requirement. LMP-1 gene was successfully acquired. The entry clone was verified by enzymes digestion, and sequencing. The constructed pAd-LMP-1 recombination adenovirus vector was successfully packaged in 293A cells. This result demonstrated that it is easy and rapid to obtain pAd-LMP-1 by constructing pAd-LMP-1 recombination adenovirus vector using the Gateway technology. Source

Weng G.,Southern Medical University | Zeng Y.,Jiangmen Wuyi Traditional Chinese Medicine Hospital | Huang J.,Shenzhen Traditional Chinese Medicine Hospital | Fan J.,Jiangmen Wuyi Traditional Chinese Medicine Hospital | Guo K.,Southern Medical University
BioMed Research International

Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CUR-And CUR+BUS-Treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin. © 2015 Guangyang Weng et al. Source

Gao L.-L.,Tianjin University of Traditional Chinese Medicine | Guo Y.,Tianjin University of Traditional Chinese Medicine | Sha T.,Tianjin University of Traditional Chinese Medicine | Liu Y.-Y.,Tianjin University of Traditional Chinese Medicine | And 5 more authors.
Acupuncture in Medicine

Objective The 'intensity-response' relationship between acupuncture stimulation and therapeutic effect is currently the focus of much research interest. The same needling manipulation with different frequencies can generate differential levels of stimulus. This study aimed to examine the effects on gastric motility induced by four twirling frequencies based on relatively standardised manual acupuncture (MA) manipulations. Methods Twirling manipulations at 1, 2, 3, and 4 Hz were practised before the experiments by a single operator using an MA parameter measurement device and stability was evaluated through time-frequency analysis. Forty-eight Sprague-Dawley rats were randomly divided into six groups (n=8 each): Control, Model, Model +MA (1, 2, 3, and 4 Hz). Rats in the five Model groups received injections of atropine into the tail vein to inhibit gastric motility, which was continuously recorded by a balloon in the gastric antrum. Rats in the four Model+MA groups received MA at 1, 2, 3 and 4 Hz, respectively, for 70 s and needles were retained for a further 5 min. Results The amplitude of waveforms produced by the four twirling frequencies was relatively consistent and reproducible. The gastric motility amplitude in all groups decreased after modelling (injections of atropine) (p<0.01). Twirling manipulation at 1, 2, and 3 Hz (but not 4 Hz) increased gastric motility amplitude (p<0.05). The increase in gastric motility amplitude induced by MA at 2 Hz was greater than for all other frequencies (p<0.05). Conclusions Acupuncture at ST36 helped recover gastric motility amplitude in rats with atropine-induced gastric inhibition and the effects induced by 1'3 Hz frequency were greater than those induced by 4 Hz. Source

Wang Y.-Y.,Shenzhen Traditional Chinese Medicine Hospital | Liao S.-H.,Peking University | Zhang S.-N.,Shenzhen Traditional Chinese Medicine Hospital
International Eye Science

AIM: To observe the influence of free iron, synthetic or biological iron chelating agent on the formation of pseudomonas aeruginosa biofilm on the contact lenses. METHODS: Different concentration of free iron, different concentration and variety classes of synthesis iron and biological iron chelating agent were added into pseudomonas aeruginosa biofilm, and then the clump count on the biofilm was detected. RESULTS: Low concentration of free iron increased the adhesive capacity of the bacterial, with 10μmol/L of iron had the most obvious effect. 0.1μmol/L of EDTA and lactoferrin significantly inhibited the bacterial count of pseudomonas aeruginosa biofilm. CONCLUSION: Low concentration of free iron can promote the bacterial count of pseudomonas aeruginosa biofilm on the contact lens, the lack iron circumstance which formed by finite concentration lactoferrin and EDTA can prevent the formation of the biofilm. Source

Zeng Y.,Sun Yat Sen University | Li S.,Shenzhen Traditional Chinese Medicine Hospital | Wu J.,Sun Yat Sen University | Chen W.,Sun Yat Sen University | And 4 more authors.

Aristolochic acid I (AAI) can induce renal tubular epithelial cells (RTECs) autophagy, which thereby extenuates apoptosis in vitro. In this study, we aimed to determine whether the in vitro data also apply to the AAI-induced pathologic condition in vivo. BALB/c mice were treated with AAI, autophagy inhibitors [3-methyladenine (3MA) or chloroquine diphosphate salt (CQ)], and AAI plus the inhibitors for consecutive 5 days, respectively. Mice were euthanized on day 3 and 5. AAI induced RTECs autophagy was confirmed by electron microscopy and western blot. The results showed induction of apoptotic RTECs and up-regulation of mitochondrial and endoplasmic reticulum stress-related proteins in AAI-treated mice at both of the two time points. There were more apoptotic RTECs in AAI + inhibitor groups, which might be due to increased mitochondrial stress-related proteins (cytochrome C and apoptotic protease activating factor 1, APAF-1). On day 5, severe tubulointerstitial injuries induced by AAI led to a significant decline in kidney function. There were numerous autolysosomes in dying RTECs of the AAI group. Autophagy inhibitors increased AAI-induced RTECs mitochondrial apoptosis by increasing mitochondrial stress-related proteins, but they partially mitigated the AAI-induced severe renal tubulointerstitial injury. These results confirmed that AAI could induce autophagy in RTECs, which prevented apoptosis via mitochondrial pathway in vivo. However, continuous stimulation with AAI induced excess autophagy, which ultimately resulted in AAI-induced cell death. It suggested that apoptosis wasn't the main culprit in acute aristolochic acid nephropathy mice model. © 2014 Springer Science+Business Media. Source

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