FDC-SP as a diagnostic and prognostic biomarker and modulates immune infiltrates in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC), one of the top 10 causes of cancer death, is responsible for more than 90% of all cases of primary renal cancer worldwide. Follicular dendritic cell-secreted protein (FDC-SP) specifically binds to activated B cells and regulates the generation of antibodies. It is also thought to promote cancer cell invasion and migration, which could help with tumor metastases. This study aimed to assess the efficacy of FDC-SP in the diagnosis and prognosis of RCC and to investigate the relationship between immune infiltration in RCC and these outcomes. RESULTS: RCC tissues had significantly higher levels of FDC-SP protein and mRNA than normal tissues. The high level of FDC-SP expression was linked to the T stage, histological grade, pathological stage, N stage, M stage, and OS event. Functional enrichment analysis identified the major pathways that were enriched as immune response regulation, complement, and coagulation. Immunological checkpoints and immune cell infiltration were observed to substantially correlate with the levels of FDC-SP expression. FDC-SP expression levels showed the ability to precisely distinguish high-grade or high-stage renal cancer (area under the curve (AUC) = 0.830, 0.722), and RCC patients with higher FDC-SP expression levels had worse prognoses. The AUC values for one-, two-, and five-year survival rates were all greater than 0.600. Moreover, the FDC-SP expression is an independent predictive biomarker of OS in RCC patients. CONCLUSION: FDC-SP may be a prospective therapeutic target in RCC as well as a possible diagnostic and prognostic biomarker associated with immune infiltration.

Engineered nanomedicines block the PD-1/PD-L1 axis for potentiated cancer immunotherapy.

Immunotherapy, in particular immune checkpoint blockade (ICB) therapy targeting the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, has remarkably revolutionized cancer treatment in the clinic. Anti-PD-1/PD-L1 therapy is designed to restore the antitumor response of cytotoxic T cells (CTLs) by blocking the interaction between PD-L1 on tumour cells and PD-1 on CTLs. Nevertheless, current anti-PD-1/PD-L1 therapy suffers from poor therapeutic outcomes in a large variety of solid tumours due to insufficient tumour specificity, severe cytotoxic effects, and the occurrence of immune resistance. In recent years, nanosized drug delivery systems (NDDSs), endowed with highly efficient tumour targeting and versatility for combination therapy, have paved a new avenue for cancer immunotherapy. In this review article, we summarized the recent advances in NDDSs for anti-PD-1/PD-L1 therapy. We then discussed the challenges and further provided perspectives to promote the clinical application of NDDS-based anti-PD-1/PD-L1 therapy.

The preliminary experience of methylene blue assisted laparoscopy in the treatment of renal parapelvic cysts.

Renal cyst is a common disease in humans and laparoscopic renal cyst decortication is the gold standard for treatment. However, specialized surgical skills are required for the treatment of renal parapelvic cysts. In this study, we describe an improved laparoscopic method for the treatment of renal parapelvic cysts involving the use of continuous infusion of methylene blue. Sixteen patients with renal parapelvic cyst were enrolled in this study. All patients underwent retrograde ureteral catheterization, with continuous perfusion of the renal pelvis using a solution of 0.2% methylene blue and saline, during laparoscopic decortication of the parapelvic cyst. In one patient, the cyst communicated with the renal collection system which was injured, but this was immediately repaired intraoperatively. All operations were successful, and none was converted to open surgery. There were no occurrences of persistent urinary fistula, bleeding, or other complications postoperatively. All patients were followed-up for 3-24 months, and results of postoperative imaging investigations revealed that all of our patients experienced either complete recovery or a greater than 50% decrease in size of the cysts. Our study demonstrates that methylene blue-assisted laparoscopic treatment is a safe, effective and practical method for the treatment of renal parapelvic cysts.

[Influence of Substrate Exposure Level on ANAMMOX Microbial Activity and Biomass].

Substrate exposure levels are vital for the growth and metabolism of ANAMMOX microorganisms, and their effects on growth characteristics of ANAMMOX sludge during the enrichment process have been rarely reported. Using two continuous flow stirred reactors and the process of a gradually developing nitrogen load, the changes in biomass and activity, as well as nitrogen removal efficiency of the reactors were investigated under high substrate exposure level culture mode (R1:effluent NH4+-N and NO2--N concentrations were 40-60 mg·L-1) and low substrate exposure level culture mode (R2:effluent NH4+-N and NO2--N concentrations were 0-20 mg·L-1). The results showed that the high substrate exposure level culture mode was more beneficial to the improvement of nitrogen removal performance of the ANAMMOX reactor. For comparison, the NLR (nitrogen load rate), which was 0.69 kg·(m3·d)-1, and the NRR (nitrogen remove rate), which was up to 0.41 kg·(m3·d)-1, was obtained in the high substrate exposure culture mode. These values were twice as high as those obtained in the low substrate exposure culture mode. Under the culture mode with high substrate exposure level, the sludge concentration (in VSS) and the total gene copy numbers of ANAMMOX reached 1805 mg·L-1 and 4.81×1012 copies, respectively, which was conducive to the rapid enrichment of ANAMMOX microorganisms. In the low substrate exposure level culture mode, ANAMMOX sludge was more active,in N/VSS, 0.27 g·(g·d)-1, which was conducive to the cultivation of ANAMMOX sludge with higher biological activity.

The correlation between affected renal function and affected renal residual volume: A retrospective outcome of laparoscopic nephron-sparing partial nephrectomy with segmental renal artery blocking-up for localized renal tumors.

Laparoscopic nephron-sparing partial nephrectomy with segmental renal artery blocking (SRPN) has been widely used in the treatment of localized renal tumors. However, the impact of ischemia-reperfusion injury (IRI) during SRPN remains controversial. This study aims to evaluate the correlation between affected renal function and affected renal volume after SRPN for localized renal tumor treatment, explore the effect of IRI on renal function after SRPN.A total of 39 patients who underwent SRPN for localized renal tumor from June 2009 to April 2012 were reviewed. These patients were followed-up for 5 years. The preoperative affected renal glomerular filtration rate (aGFRpre), postoperative affected renal glomerular filtration rate (aGFRpost), preoperative affected renal volume (aVolpre), and postoperative affected renal volume (aVolpost) were collected during the follow-up period. The correlation between aGFRpost/aGFRpre and aVolpost/aVolpre was compared.A total of 33 patients were successfully followed up. After 3, 6, 12, 24, and 60 months, aGFRpost was 34.6 ±â€Š4.6, 34.7 ±â€Š4.8, 34.9 ±â€Š4.4, 35.1 ±â€Š4.4, and 35.2 ±â€Š4.2 mL/min. The correlation coefficients between aGFRpost/aGFRpre and aVolpost/aVolpre were 0.659 (P = .000), 0.667 (P = .000), 0.663 (P = .000), 0.629 (P = .000), and 0.604 (P = .000), respectively. The limitation of this study was the small cohort size.For the localized renal tumor, aGFRpost was associated with aVolpost, but was not associated with intraoperative factors, such as the time of clamping of the affected segmental renal artery. As a part of nephrons, the resected tumor tissue caused the lack of inherent nephrons, resulting in the loss of renal function. More nephrons should be maintained before resecting the tumor completely during SRPN.Trial registration: ChiCTR-RRC-17011418.

A New Technique: Laparoscopic Resection of Fibrous Ring and Placing Extravascular Stent in Patients With Nutcracker Syndrome: A Report of 5 Cases.

OBJECTIVE: To evaluate the feasibility and safety of a new laparoscopic technique for resection of a fibrous ring and extravascular stent implantation in patients with nutcracker syndrome (NCS). MATERIAL AND METHODS: We retrospectively reviewed data of 5 patients diagnosed with NCS between March 2010 and February 2016. The mean age of the patients (4 male and 1 female) was 34 years (range, 28-40 years). All 5 patients underwent laparoscopic resection of the narrow fibrous ring around the left renal venous (LRV) and for extravascular stent implantation in the LRV for management of NCS. RESULTS: The average operating time was104 minutes and the average blood loss during surgery was 59 mL. The average length of the postoperative hospital stay was 6 days (range, 4-8 days). In all 5 patients, the symptoms of macroscopic hematuria started decreasing gradually and resolved after surgery. Postoperative computed tomography showed that the blood outflow from the LRV was smooth. The ratio of the dilated segment's inner diameter to the diameter of the strictured segment decreased from 3.4 to 9.5, preoperatively to 1.1-2.0, postoperatively. The mean follow-up period was 17.6 months (range, 8-24 months).One patient's varicocele was cured and symptoms in all 5 patients resolved after surgery. None of the patients showed symptom recurrence. CONCLUSION: Laparoscopic surgery, for the placement of an extravascular stent and resection of the fibrous ring around the end of the LRV outflow to the inferior vena cava appears feasible and safe and offers an alternative minimally invasive for the management of NCS.

[Effect of Temperature and pH on Nitrogen Conversion in Feammox Process].

In recent years, the oxidation of NH4+ using Fe(Ⅲ) as an electron acceptor under anaerobic conditions (Feammox) has received significant research attention. In this study, the effect of pH and temperature on nitrogen conversion during the Feammox process was studied through activity recovery of Feammox sludge acclimated by anaerobic ammonium oxidation (ANAMMOX) sludge. Results showed that after 40 d operation, activity of Feammox sludge was recovered. There was evident ammonia nitrogen conversion and total nitrogen removal from the environment, and the products were mainly nitrate and nitrogen. The concentration of nitrite remained below 2 mg·L-1. pH value and temperature significantly influenced nitrogen transformation during the Feammox process. With pH value of 7 and temperature of 30℃ during the Feammox process, the removal rate of total nitrogen was relatively high (>50%). When the pH value was 6.5, the conversion rate of ammonia nitrogen was 80.2%. During the Feammox reaction process, precipitation of iron ion compounds and coating on the sludge surface were the main interference factors leading to continuous operation of the reactor and exploration of the reaction mechanism.

CD164 promotes tumor progression and predicts the poor prognosis of bladder cancer.

CD164 was found to play a role in many malignant diseases. But the roles of CD164 in human bladder cancer have not yet been studied. The object of our study was to investigate the functions of CD164 in urothelial bladder carcinoma. The immunohistochemistry (IHC) was performed to evaluate the associations between the expression level of CD164 and clinical-pathological features of patients, and IHC was used to analyze the relationship between CD164 and CXCR4 in tumor tissues. Real-time qPCR and Western blot were used to measure the expression of relevant genes. The roles of CD164 in tumor cells and tissues were investigated by in vitro and in vivo experiments. The results of immunohistochemistry found that CD164 was associated with clinical and pathological features of patients. High level of CD164 was related to the distant metastasis and vascular invasion of bladder cancer patients. In vitro, by silencing of CD164, the proliferation, migration, and invasion of tumor cells were inhibited significantly by regulating related proteins such as Ki67, proliferating cell nuclear antigen, matrix metalloproteinases-2, and matrix metalloproteinases-9. In vivo, knocking-down of CD164 could reduce the growth and metastasis of tumors in mice. In addition, a co-expression was found between CD164 and CXCR4 in tumor tissues. In conclusion, our study demonstrated that CD164 was associated with the poor clinical outcomes of BC patients. Silencing of CD164 could inhibit the progression of tumors in vivo and in vitro, which may become an effective target in the treatment of bladder cancer.

[Analysis of CANON Process Start-up with Fiber Carrier].

A CANON reactor with fiber carrier was started up by seeding nitrification sludge and ANAMMOX sludge to study the operating characteristics of a fiber carrier. The results showed that total nitrogen removal load rose from 0.09 kg·(m3·d)-1 to 0.9 kg·(m3·d)-1 and remained steady in the 85th day. This indicated that fiber carrier is beneficial to the accumulation of sludge, and the reactor can maintain a higher biomass. The DO in the reactor reached 5 mg·L-1 with the enrichment of microorganisms, biofilm thickening, and the improvement of the reactor's ability. The DO gradient of the biofilm from the outside to the inside was 0.32-0 mg·L-1, which could be obtained by a microelectrode. It was shown that the permeability of oxygen to the biofilm decreased, and the amount of nitrifying microorganisms decreased with biofilm thickening. The quantitative PCR results showed that the abundance of ANAMMOX was an order of magnitude more than before. The abundance of AOB increased slightly, while the abundance of NOB stayed at a relatively low level.

[Inhibitory Effects of Phosphate and Recovery on a Nitrification System].

The effect of phosphate concentration on nitrification was studied by using a stabilization nitrosation system, which was started up in a continuous flow reactor by inoculating sludge from a municipal wastewater treatment plant. The results showed that the nitrification system was started successfully after operating for 14 days. The conversion rate of ammonia nitrogen reached 92.2%, the nitrite accumulation rate was 73.66%, and the nitrite generation rate was 14.42 g·(m3·d)-1. There was no effect of phosphate concentration on the nitrosation system between 10 and 30 mg·L-1; and the conversion rate of ammonia nitrogen was decreased with the continuous increase in phosphate concentration. When the concentration of phosphate was 80 mg·L-1, with an ammonia conversion rate 13.6%, accumulation rate of nitrite of 18.19%, and nitrite generation rate of 0.54 g·(m3·d)-1, the reaction was severely inhibited. After reducing the influent phosphate concentration to 0, with the ammonia nitrogen conversion rate at more than 80%, nitrite accumulation rate improved to 86.96%, and the nitrite generation rate being 15.63 g·(m3·d)-1, the system recovered after operating for 14 days.

[Granular Characteristics of Anaerobic Ammonia Oxidation Sludge During the Recovery Process].

Operation instability has become one of the factors restricting the application of the anaerobic ammonia oxidation (ANAMMOX) process. Under the condition that the substrate is not suppressed, the effects of the substrate concentration on the granulation and activity of ANAMMOX granular sludge in the recovery process were studied by restoring the activity of ANAMMOX sludge, which was derived from early-stage operation instability of the continuous stirred tank reactor (CSTR). The results show that the activity of ANAMMOX sludge was recovered and the denitrification capacity increased significantly after 126 days of operation. When the NH4+-N and NO2--N concentrations were 450 mg·L-1 and 560 mg·L-1, respectively, the nitrogen removal was achieved in both the high-and low-substrate concentration reactors and the maximum NRR was 16.97 kg·(m3·d)-1 and 14.43 kg·(m3·d)-1, respectively. With the improvement of the nitrogen removal capacity of the reactor (the granular diameter of the sludge is increased), the extracellular polymeric substance (EPS) content increased in both reactors from 34.45 to 77.52 and to 94.18 mg·g-1, respectively, and the PN/PS increased from 1.89 to 6.25 and 6.84, respectively. To a certain extent, the increase of PN/PS is conducive to the granulation of ANAMMOX sludge, but a too large PN/PS would lead to the instability of granular sludge and sludge loss.

Therapy and Prevention of Postoperative Urosepsis of Ureter Endoscopic Lithotripsy for Non-infection.

Objective To analyze the risk factors causing postoperative urosepsis in ureter endoscopic lithotripsy without infection preoperatively, in order to make a more effective and safer preventive and therapeutic strategy.Methods From January 2010 to January 2015, 5 ureteral calculus patients undergoing ureter endoscopic lithotripsy with holmium laser were retrospectively enrolled in this clinical study. These patients suffered urosepsis postoperatively confirmed by the clinical presentations and laboratory Results, while they had no infection in their blood and urine preoperatively. Without delay, 5 patients were treated by anti-inflammation and anti-shock.Results The vasopressor drug was stopped gradually after 12-36 hours. The body temperature was recovered to normal in 2 or 3 days, and the blood and urine test Results were not abnormal in 7 days. At last, 5 patients were all cured.Conclusions Stone and operation themselves are potential factors to cause urosepsis after ureter endoscopic lithotripsy. Especially for patients who had not presented infection preoperatively, careful preparation preoperatively, corrective manipulation, low pressure irrigation, drainage and controlling time during operation, and early diagnosis, appropriate treatment postoperatively are the key to cure and prevent urosepsis.

Neuroprotective effect of methylprednisolone combined with placenta-derived mesenchymal stem cell in rabbit model of spinal cord injury.

The aim of this study was to assess the ability of the combination treatment of methylprednisolone (MP) and placenta-derived mesenchymal stem cells (PDMSCs) in a rabbit model of spinal cord injury (SCI). Rabbits were randomly divided into four groups: group 1 (control), group 2 (MP), group 3 (PDMSCs) and group 4 (MP + PDMSCs). In all groups, the spinal cord injury model was created by the weight drop method. Levels of malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were determined by kit. Histopathological examination was also performed. Neurological evaluation was carried out with the Tarlov scoring system. The results showed both MP and PDMSCs had neuroprotective effects, and combining the administration of MP with PDMSCs was shown a significant effect on the recovery of neurological function. Therefore, the combined use of MP and PDMSCs can be used as a potential therapeutic method for SCI.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons (sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteogenic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the proliferation of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-related factors (including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which provides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

[Influence and distribution of CD133 and CD34 antigen expression in proximal tubule epithelial cells during ischemia/reperfusion injury in rats].

OBJECTIVE: To study the influence and distribution in proximal tubule epithelial cells with the expressions of CD133 and CD34 in a rat model so as to provide a study basis for renal adult stem cell. METHODS: The kidney ischemia/reperfusion model was established by blocking the bilateral renal arteries for about 50 min and recovering the renal perfusion of blood. Then the rat kidneys were extracted at Days 3, 5 and 7 post-modeling. After a series of special treatment, immunohistochemical staining was used to show the distribution and expression intensity of KIM-1, Brdu antigen, CD34 and CD133 antigens in cells with the elapsing of time. RESULTS: As compared with control group, the KIM-1 and CD133 antigens were present in cortex renis while the CD34 and Brdu antigens were distributed in parts of medulla renis and juncture of cortex-medulla renis. The expression density value of KIM-1 and CD133 antigens rose for the first 3 days then declined afterward (40.3% ± 3.2%, 57.5% ± 3.8%, 24.3% ± 1.4%). Otherwise the expression density value of CD 34 antigen declined (56.0% ± 4.8%, 44.2% ± 2.2%, 28.8% ± 1.0%) and Brdu antigen showed an upward trend at post-operation (10.0% ± 1.1%, 36.0% ± 4.2%, 48.8% ± 5.0%). CONCLUSION: After ischemia/reperfusion injury in kidney, the expression rates of CD133 and KIM-1 antigen rise obviously in cortex renis. And the D34 and Brdu antigens show a similar trend in medulla renis. The result indicates that the phenomenon of proliferation and differentiation may appears in kidney proximal tubule and migrate from the region of medulla renis to cortex renis. The participating cells not only possess the strong proliferation and repairing ability of stem/progenitor cells, but also can expresses the CD34 and CD133 antigens. Thus it is may provide a study basis for the tissue reconstruction of nephron and research in the field of kidney adult stem cell.

Apoptosis-inducing effects of curcumin derivatives in human bladder cancer cells.

Our aim was to prepare curcumin derivatives and study their apoptosis-inducing effects on bladder cancer cells in order to establish a basis for targeted chemotherapy of cancer. n-Maleoyl-L-valine-curcumin (NVC) and n-maleoyl-glycine-curcumin (NGC) were chemically synthesized. Intracellular esterase activity of the human bladder cancer EJ cell line and renal tubular epithelial (HKC) cells was examined by 6-carboxyfluorescein diacetate fluorometry. After incubation with NVC or NGC for 6-24 h, cell viability was detected by MTT colorimetry. Cell apoptosis and apoptotic rates were measured by acridine orange/ethidium bromide staining, TUNEL labeling and flow cytometry. Intracellular caspase-3 activities were determined by spectrophotometry. The esterase activity within EJ cells was 10.2-fold higher than that of HKC cells, which was abolished by bis-p-nitrophenylphosphate, an esterase inhibitor, resulting in decreases in NVC- and NGC-mediated cell viability arrest. For EJ cells, the IC50 values of NVC (20.1 micromol/l) and NGC (18.7 micromol/l) were close to curcumin (16.5 micromol/l). Meanwhile, their IC50 values on HKC cells were, respectively, 4.06- and 3.23-fold higher than curcumin. Moreover, NVC and NGC induced apoptosis of EJ cells by 10.13-23.36 and 12.42-28.56%, respectively. Administration of these two derivatives resulted in decreased apoptosis of HKC cells compared with curcumin. The caspase-3 activities of EJ cells, but not of HKC cells, were 5.21- and 5.63-fold enhanced by NVC and NGC, respectively. Thus, novel esterase-sensitive curcumin derivatives were synthesized, which induced extensive apoptosis of bladder cancer EJ cells, but not normal cells.

[Effects of recipes for replenishing qi and activating blood on senescence related gene expressions in the liver of aging rats].

OBJECTIVE: To observe the effects of recipes for replenishing qi and activating blood on p16, p21, proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E gene expressions in the liver of aging rats. METHODS: A recipe for replenishing qi and a recipe for activating blood were administered to aging rats respectively, and the effects of the above recipes on the expressions of senescence related genes (p16, p21, PCNA, cyclin D1 and cyclin E) were examined by RT-PCR and Western blotting methods. RESULTS: The expressions of p16, p21 and cyclin D1 mRNAs and proteins in the liver of the untreated aging rats were up-regulated, while the expressions of PCNA and cyclin E mRNAs and proteins decreased. As compared with the untreated aging rats, both recipes could down-regulate the expressions of cyclin D1 mRNA and protein and up-regulate the expressions of cyclin E mRNA and protein, but had no obvious effects on the expressions of mRNAs and proteins of p16, p21 and PCNA. CONCLUSION: Recipes for replenishing qi and activating blood can improve the liver cell proliferation of aging rats via down-regulating the expressions of cyclin D1 mRNA and protein and up-regulating the expressions of cyclin E mRNA and protein.

Downregulation of XIAP expression induces apoptosis and enhances chemotherapeutic sensitivity in human gastric cancer cells.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.

Selection of optimal antisense accessible sites of survivin and its application in treatment of gastric cancer.

AIM: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer. METHODS: The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT) colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry. RESULTS: Thirteen AAS of survivin were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with non-tranfection controls, their corresponding AS-ODNs (AS-ODN(1), AS-ODN(2), AS-ODN(3) and AS-ODN(4)) could reduce Survivin mRNA levels in MKN-45 cells by 54.3+/-1.1% (t = 6.12, P<0.01), 86.1+/-1.0% (t = 5.27, P<0.01), 32.2+/-1.3% (t = 7.34, P<0.01) and 56.2+/-0.9% (t = 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2+/-2.5% (t = 6.26, P<0.01), 75.4+/-3.1% (t = 7.11, P<0.01), 28.3+/-2.0% (t = 6.04, P<0.01) and 45.8+/-1.2% (t = 6.38, P<0.01) respectively. After transfection with 600 nmol/L AS-ODN1-AS-ODN(4) for 24 h, cell growth was inhibited by 28.12+/-1.54% (t = 7.62, P<0.01), 38.42+/-3.12% (t = 7.75, P<0.01), 21.46+/-2.63% (t = 5.94, P<0.01) and 32.12+/-1.77% (t = 6.17, P<0.01) respectively. Partial cancer cells presented the characteristic morphological changes of apoptosis, with apoptotic rates being 19.31+/-1.16% (t = 7.16, P<0.01), 29.24+/-1.94% (t = 8.15, P<0.01), 11.87+/-0.68% (t = 6.68, P<0.01) and 21.68+/-2.14% (t = 7.53, P<0.01) respectively. CONCLUSION: The AAS of survivin could be effectively selected in vitro by random oligonucleotide library/RNase H cleavage method combined with computer software analysis, this has important reference values for further studying survivin-targeted therapy strategies for gastric cancer.

[Comparative study on the regulatory effects on senescence related cell cycle gene expression by TCM principles of tonifying shen, invigorating pi benefiting qi, and activating blood circulation].

OBJECTIVE: To compare the effects of TCM therapeutic principles of tonifying Shen (TS), benefiting Qi (BQ), invigorating Pi (IP) and activating blood circulation (ABC) herbs in regulating the gene expression in senescence related cell cycle. METHODS: Drug sera containing TCM herbs of the above-mentioned principles were used to treat the aged human diploid fibroblast cell line 2BS. The effect of TCM on the senescence related cell cycle and its related gene expression (P16INK4, Cyclin D1 and PCNA) were examined by means of cell proliferative doublings, flow cytometry, RT-PCR and Western blot analysis. RESULTS: TCM herbs of TS and BQ could improve the cell cycle, down-regulate the P16 and Cyclin D1 mRNA/protein expression, up-regulate PCNA mRNA/protein expression, while TCM herbs of IP and ABC showed insignificant effect on these indexes. CONCLUSION: TCM herbs of TS and BQ have effect in improving cell cycle, it may be achieved through promoting the P16 pathway of gene expression.