Infected bone resection plus adjuvant antibiotic-impregnated calcium sulfate versus infected bone resection alone in the treatment of diabetic forefoot osteomyelitis.

BACKGROUND: Managing with diabetic foot osteomyelitis (DFO) is challenging. Even after infective bone resection and thorough debridement, DFO is still difficult to cure and has a high recurrence rate. This retrospective study aims to compare the outcomes of two treatment methods, infected bone resection combined with adjuvant antibiotic-impregnated calcium sulfate and infected bone resection alone, for the treatment of diabetic foot osteomyelitis. METHODS: Between 2015 to 2017, 48 limbs (46 patients) with DFO met the criteria were included for assessment. 20 limbs (18 patients) were included in the calcium sulfate group (the CS group) in which vancomycin and/or gentamicin-impregnated calcium sulfate was used as an adjuvant after infected bone resection while 28 limbs (28 patients) as the control group were undergone infected bone resection only. Systemic antibiotics, postoperative wound care and offloading were continued to be applied following surgery in both groups. The time to healing, healing rate, recurrence rate and amputation rate were compared between the two groups. RESULTS: In total, 90% (18/20) limbs in the CS group as compared to 78.6% (22/28) infected limbs in the control group went to heal (P = 0.513). The Mean time to healing was 13.3 weeks in the CS group and 11.2 weeks in control group (P = 0.132). Osteomyelitis recurrence rate was 0% (0/18) in the CS group and 36.4% (8/22) in the control group (P = 0.014). Postoperative leakage in calcium sulfate group was 30.0% (6/20) with a mean duration of 8.5 weeks. Amputation rate in the control group was 7.1% (2/28) compared to 0% (0/20) in the CS group (P = 0.153). CONCLUSIONS: Antibiotic-impregnated calcium sulfate as an adjuvant prevents the recurrence of DFO but cannot improve the healing rate, reduce the postoperative amputation rate or shorten the time to healing. Prolonged postoperative leakage as the most common complication can be managed with regular dressing. LEVEL OF EVIDENCE: III, Retrospective Comparative Study.

One-stage debridement and bone transport versus first-stage debridement and second-stage bone transport for the management of lower limb post-traumatic osteomyelitis.

BACKGROUND: Treatment of lower limb post-traumatic osteomyelitis used to be a staged process, with radical debridement of bone and soft tissues at first stage, followed by a second-stage limb reconstruction operation to restore the limb integrity. Some studies recently reported that achieving infection eradication and limb reconstruction at single-stage seems to be an effective method for lower limb infection, but a comparative study remains lacking. This study aims to compare the results of radical debridement combined with a first/second-staged osteotomy and bone transport, for the management of lower limb post-traumatic osteomyelitis. METHODS: From January 2013 to June 2018, a total of 102 patients with lower limb post-traumatic osteomyelitis met the criteria were included for analysis, in which 70 patients received one-stage debridement, antibiotic-loaded implantation, metaphysis osteotomy and bone transport were named as one-stage group, while 32 patients with first-stage debridement and antibiotic-loaded calcium sulfate implantation, second-stage osteotomy and bone transport were devised as two-stage group. The outcomes of hospitalization (hospital stay, costs of treatment, surgical time, antibiotic usage) and follow-up (infection-free, treatment failure, infection recurrence, external fixation index (EFI) and docking site union) between the two groups were retrospectively compared. RESULTS: For outcomes of hospitalization, patients in the one-stage group had batter results on hospital stay (18.2 days versus 28.9 days, P â€‹< â€‹0.05), surgical time (164.8 â€‹min versus 257.4 â€‹min, P â€‹< â€‹0.05), cost of treatment (¥101726.1 versus ¥126718.8, P â€‹< â€‹0.05) and the course of antibiotic usage (10.3 days versus 12.0 days, P â€‹< â€‹0.05). During the follow-up, 87.1% (61/70) patients in the one-stage group compared to 93.8% (30/32) patients in the two-stage group achieved infection-free (P â€‹> â€‹0.05) without any additional debridement operation. 94.3% (66/70) patients in the one-stage group earned wound healing after the operation, comparing to 96.9% (31/32) patients healed in the two-stage group (P â€‹> â€‹0.05). Uncontrolled infection was observed on 4 (5.7%) patients in the one-stage group and 1 (3.1%) patients in the two-stage group (P â€‹> â€‹0.05), with a result of three achieved infection free in the one-stage group and one patient suffered from amputation in each group respectively. 5 (7.2%) patients in the one-stage group and 1 (3.2%) patient in the two-stage group encountered with infection recurrence (P â€‹> â€‹0.05) and were well-managed with re-debridement and antibiotics usage. Significance was not found between two groups on EFI (74.8 days/cm versus 69.0 days/cm, P â€‹> â€‹0.05) and docking site nonunion rate (14.5% versus 18.9%, P â€‹> â€‹0.05), indicating that bone transport in different stages played a less essential role on bone generation process. The other complications, such as prolonged aseptic drainage [24.3% (17/70) versus 21.9% (7/32)], re-fracture [5.8% (4/69) versus 3.2% (1/31)], pin-tract infection [23.2% (16/69) versus 19.4% (6/31)], joint stiffness and deformity [26.1% (18/69) versus 32.3% (10/31)], also showed less significance when comparing between two groups (P â€‹> â€‹0.05), suggesting that different transport stages play little role on complications formation. CONCLUSIONS: One-stage radical debridement and bone transport was proven to be a safe and effective method for treating static (or near static) lower limb osteomyelitis. TRANSLATIONAL POTENTIAL STATEMENT: Translational potential statement One-stage debridement and bone transport is sample, effective and time-saving, with similar complications compared to conventional two-stage protocol. This treatment protocol might provide an alternative for the treatment of static (or near static) lower limb osteomyelitis.

Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine.

Whole tumor cell lysates (TCL) have been implemented as tumor antigens for cancer vaccine development, although clinical outcomes of TCL-based antitumor immunotherapy remain unsatisfactory. In order to improve the efficacy of TCL-based vaccines, biomaterials have been employed to enhance antigen delivery and presentation. Here, we have developed chitosan nanoparticles (CTS NPs) with surface mannose (Man) moieties for specific dendritic cells (DCs) targeting (Man-CTS NPs). The Man-CTS NPs were then loaded with TCL generated from B16 melanoma cells (Man-CTS-TCL NPs) for in vitro and in vivo assessment. Potency of the Man-CTS-TCL NPs as cancer vaccine was also assessed in vivo by immunization of mice with Man-CTS-TCL NPs followed by re-challenge with B16 melanoma cell inoculation. We have shown here that Man-CTS-TCL NPs promote bone marrow-derived dendritic cells (BMDCs) maturation and antigen presentation in vitro. In vivo evaluation further demonstrated that the Man-CTS-TCL NPs were readily taken up by endogenous DCs within the draining lymph node (DLN) following subcutaneous administration accompanied by increasing in serum IFN-γ and IL-4 levels. Tumor growth was also significantly delayed in mice primed with Man-CTS-TCL NPs vaccine, attributable at least in part to cytotoxic T lymphocytes response. Moreover, Man-CTS-TCL NPs vaccine also exhibited therapeutic effects in mice with melanoma. Thus, we report here the Man-CTS-TCL NPs as effective anti-tumor vaccine for cancer immunotherapy.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells.

AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector. METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001). CONCLUSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Periosteal osteosarcoma and Marfan's syndrome: A case report and literature review.

Periosteal osteosarcoma (POS) is a rare primary malignant bone tumor arising from the surface of long bones. In addition, Marfan's syndrome (MFS) is an infrequent hereditary autosomal dominant connective tissue disorder with high penetrance and variable phenotypes, which primarily affects the ocular, skeletal and cardiovascular systems. The present study reported a case of POS and MFS co-occurring in a child. A 6-year-old girl with MFS presented with pain, swelling and deformity in the right thigh following a fall. The patient was diagnosed with a right femoral shaft fracture and underwent open internal fixation surgery at a local hospital. At 2 weeks following surgery, the patient's parents observed increased swelling in the right thigh and thus, revisited the clinic. X-ray examination revealed extensive osteotylus around the fracture site and the clinician decided to remove the internal fixation. Following removal of the implant, aggravated swelling and superficial venous engorgement were observed. The patient was then admitted to Nanfang Hospital, where magnetic resonance imaging was performed, which identified symptoms of an abnormal periosteal reaction with bone erosion, indicating POS. The patient underwent a wide resection of the tumor and the histopathological examination confirmed the diagnosis of POS. No recurrence was identified at 9 months postoperatively. In conclusion, the present case report may result in increased awareness of the possibility of malignant bone tumors in a hereditary patient with osteotylus overgrowth following fracture surgery; in addition, the present case indicated a possible correlation between POS and MFS.

Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells.

AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR-CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured. RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.

[Effect of KDR recombinant adenovirus containing double suicide gene on proliferation, apoptosis and cell cycle of human umbilical vein endothelial ECV304 cells].

OBJECTIVE: To study the effect of adenovirus (Ad)-mediated fusion gene systemdriven by KDR promoter on the proliferation, apoptosis and cell cycle of human umbilical vein endothelial ECV304 cells. METHODS: The KDR-expressing ECV304 cells and LS174T cells not expressing KDR were both infected by the AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-flurocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The killing effects of the transfection on the cells were evaluated and bystander effects analyzed by coculturing the uninfected cells by AdKDR-CDglyTK with different ratios of infected cells. Flow cytometry was employed for determining the cell cycle distribution and electron microscopy performed to observe the pathological changes of cells. RESULTS: The infection rates of the resultant recombinant Ad (rAd) were similar in the cells and gradually increased with the increment in the multiplicity of infection (MOI) of the Ads. The infected cells exhibited different sensitivities to the two prodrugs: ECV304 cells infected with rAd were highly sensitive to the prodrugs, but the infected LS174T cells were not (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stronger than that of either single suicide gene (P<0.001), showing also obvious bystander effect. In addition, the cell cycle of ECV304 cells was arrested at S phase with morphologic features of apoptosis and necrosis as displayed by electron microscopy. CONCLUSIONS: CD/TK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK-expressing endothelial cells, the mechanism of which may involve cell cycle arrest and necrosis and apoptosis of the cells.

[Specific killing effects of adenovirus-mediated double suicide gene driven by KDR promoter on venous endothelial cells and gastric cancer cells in vitro].

OBJECTIVE: To study the specific killing effect of adenovirus(Ad)-mediated double suicide gene under regulation by KDR promoter on gastric cancer cells and venous endothelial cells in vitro. METHODS: KDR-expressing MGC803 and ECV304 cells and non-KDR-expressing LS174T cells were infected by the two Ads (AdEasy-KDR-CDglyTK and AdEasy-CMV- CDglyTK), and expression of CDglyTK was detected by reverse transcriptional (RT) PCR. After treatment with 5-FC and GCV, the killing effects of the double suicide genes on various cells were evaluated. RESULTS: The infection rate of the two resultant recombinant Ads did not differ significantly in the cells. RT-PCR demonstrated the presence of CDglyTK gene product in all the cells infected by Ad-CMV-CDglyTK and all but SL147T cells infected by Ad-KDR-CDglyTK. All the cells infected by Ad-CMV-CDglyTK and ECV304 and MGC803 cells infected Ad-KDR-CDglyTK were highly sensitive to the prodrugs. In contrast, LS174T cells infected by Ad-KDR-CDglyTK did not appear sensitive to the two prodrugs (P<0.001). In addition, the effect of the double suicide gene was much stronger than that of either of the single suicide gene (P<0.001), showing also considerable bystander effect. CONCLUSIONS: The double suicide gene driven by KDR promoter has specific killing effect on KDR-expressing gastric tumor cells and venous endothelial cells in vitro.

[Clinical observation of abdominal compartment syndrome: report of 11 cases].

Through clinical observation of 11 patients with abdominal compartment syndrome (ACS), the author explored the effects of acutely elevated abdominal pressure on systemic circulation, respiration and renal function. Based on the experience in diagnosis and treatment of ACS, the author suggests that early identification and timely surgical depressurization are essential to reduce mortality and improve the prognosis.

Dynamic changes in serum level of nitric oxide and its mechanisms in rats with traumatic shock.

OBJECTIVE: To investigate the dynamic changes of serum nitric oxide (NO) level and its mechanisms in rats in traumatic shock. METHODS: Rat models of traumatic shock were established by fracturing the posterior limb of the rats. Serum levels of nitrate/nitrite were measured with the method described by Griess. RESULTS: No significant changes in serum NO level took place during traumatic shock. One hour after resuscitation, however, serum NO level first underwent marked decline and then increased to the peak level at 6 h after resuscitation, retaining a high level till 24 h after resuscitation. No obvious changes of serum NO levels were observed in rats without resuscitation. CONCLUSION: Serum NO level does not significantly alter during traumatic shock, while after resuscitation, NO level increases after transient decrease, possibly due to reperfusion injury.

[Adenovirus-mediated CDglyTK fusion gene system driven by KDR promoter selectively kills MCF-7 breast cancer cells and vascular endothelial cells].

OBJECTIVE: To observe the selective killing of MCF-7 human breast cancer cells and vascular endothelial ECV304 cells by adenovirus (Ad)-mediated double suicide gene driven by KDR promoter. METHODS: The plasmid pAdEasy-KDR- CDglyTK was transfected into 293 packaging cells for amplification of the infectious Ad, which were then used to infect KDR-producing ECV304 and MCF-7 cells and LS174T cells that did not produce KDR. The infected cells were treated with 5-FC and/or GCV at different doses to observe the cell-killing and bystander effects of AdKDR-CdglyTK. The cell cycle changes were also detected by flow cytometry. RESULTS: The Ad at the titer of 2.0 x 10(12) pfu/ml was obtained after the amplification, whose infection rates of the cells were similar, but could be increased gradually with the multiplicity of infection (MOI) till reaching 100% with the MOI of 200. The infected cells exhibited different sensitivities to the two prodrugs: ECV304 cells and MCF-7 cells infected with Ad-KDR-CDglyTK showed similar high sensitivity to the prodrugs (P=1.00), whereas the infected LS174T cells appeared to be less sensitive (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stranger than that of either suicide gene (P<0.001), but all exhbiting considerable bystander effect. In addition, the cell cycle of MCF-7 cells was arrested at G1 phase. CONCLUSIONS: CD/TK fusion gene system driven by KDR promoter can selectively kill KDR-expressing vascular endothelial cells and MCF-7 cells.

[Therapeutic effect of L-arginine on traumatic shock in rats].

OBJECTIVE: To evaluate the therapeutic effect of L-arginine (L-Arg) on traumatic shock in rats and explore the possible mechanisms. METHODS: Rat models of traumatic shock were established in Sprague-Daulay rats, which were randomly divided into two groups either to receive L-Arg treatment or not. The plasma concentration of endothelin (ET) and oxygen partial pressure in the tissues from the skeletal muscles, liver and small intestine were measured before and after the shock and 1, 3, and 5 h after resuscitation. The hemodynamics of the rats and their survival rates at 12 and 24 h were recorded. RESULTS: The changes of plasma ET levels and oxygen partial pressure in tissues of both groups were statistically significant after traumatic shock (P<0.05). Plasma ET concentration at 5 h after resuscitation was significantly lower in the treatment group than in the non-treatment shock group (P<0.05), while oxygen partial pressure in the liver and small intestine after resuscitation were significantly higher in the treatment group (P<0.05). The survival rates at 12 and 24 h were also significantly different between the 2 groups. CONCLUSION: Adscititious L-Arg can decrease plasma ET levels, improve oxygen partial pressure of internal organs and significantly increase the survival rate of the rats with traumatic shock.

[Study on the relationship between changes of adrenomedulin in plasma and vascular resistance during traumatic shock in rats].

OBJECTIVE: To observe the changes of adrenomedulin in plasma and vascular resistance during traumatic shock in rats. METHODS: The concentration of adrenomedulin in plasma of rats after traumatic shock was detected by radio immunization. Mean arterial pressure, total peripheral vascular resistance (TPVR) and cardiac index (CI) were estimated by electrical conductance method. RESULTS: The concentration of adrenomedulin in plasma of traumatic shock without resuscitation group (68.34+/-3.71)ng/L and traumatic shock with resuscitation group (146.27+/-9.83)ng/L were higher than that of control group(32.63+/-7.55)ng/L (P<0.01 and P<0.05). TPVR of traumatic shock with resuscitation group (10.57+/-0.35) kPa.s.L-1 was lower than that of traumatic shock without resuscitation group (16.75+/-0.23) kPa.s.L-1(P<0.01) and its CI (215.59+/-1.29) ml.min-1.kg-1 was higher than that of traumatic shock without resuscitation group (143.11+/-0.86) ml.min-1.kg-1 (P<0.01). CONCLUSION: Adrenomedulin is closely correlated with changes of vascular resistance and plays an important role during pathophysiological processes in rats after traumatic shock.

The cell surface GRP78 facilitates the invasion of hepatocellular carcinoma cells.

Invasion is a major characteristic of hepatocellular carcinoma and one of the main causes of refractory to treatment. We have previously reported that GRP78 promotes the invasion of hepatocellular carcinoma although the mechanism underlying this change remains uncertain. In this paper, we explored the role of the cell surface GRP78 in the regulation of cancer cell invasion in hepatocellular carcinoma cells. We found that neutralization of the endogenous cell surface GRP78 with the anti-GRP78 antibody inhibited the adhesion and invasion in hepatocellular carcinoma cell lines Mahlavu and SMMC7721. However, forced expression of the cell surface GRP78 facilitated the adhesion and invasion in SMMC7721. We further demonstrated that inhibition of the endogenous cell surface GRP78 specifically inhibited the secretion and activity of MMP-2 but did not affect the secretion and activity of MMP-9. We also found that inhibition of the cell surface GRP78 increased E-Cadherin expression and decreased N-Cadherin level. On the contrary, forced expression of the cell surface GRP78 increased N-Cadherin expression and decreased E-Cadherin level, suggesting that the cell surface GRP78 plays critical role in the regulation of EMT process. These findings suggest that the cell surface GRP78 plays a stimulatory role in the invasion process and may be a potential anti-invasion target for the treatment of hepatocellular carcinoma.

[Prokaryotic expression, purification and antigenicity identification of human GRP78 protein].

AIM: To construct a recombinant plasmid pGEX-4T-1-GRP78, express it and purify human glucose regulated protein 78 (GRP78). METHODS: GRP78 gene was amplified by PCR from the recombinant vector constructed in our laboratory. The PCR product was inserted into pGEX-4T-1 prokaryotic expression vector to generate pGEX-4T-1-GRP78. The pGEX-4T-1-GRP78 was then transformed into BL21 and GRP78 was expressed on induction of IPTG. After purification, GRP78 was released by thrombin cleavage, and its antigenicity was identified by ELISA. RESULTS: The GRP78 prokaryotic expression vector was successfully constructed as confirmed by enzyme digestion and DNA sequencing. ELISA demonstrated the antigenicity of the purified GRP78 protein. CONCLUSION: The recombinant prokaryotic expression vector pGEX-4T-1-GRP78 has been constructed successfully. The purified GRP78 has been obtained with good antigenicity, which will be used for GRP78-based serum diagnosis of hepatocellular carcinoma.

[Identification of peptide mimotopes of an abroad-spectrum neutralizing epitope of highly pathogenic avian influenza hemagglutinin].

A monoclonal antibody (8H5), which showed strong neutralization activity against 33 strains of H5N1 viruses isolated from hosts at various regions from 2002 to 2006, was characterized in our lab recently. This result indicated the presence of highly conserved neutralizing site on hemagglutinin (HA) of various H5N1 subtypes. In the present study, the peptide phage display technique was applied to generate mimotope of the conserved neutralizing epitope recognized by 8H5 mAb. Five peptides displayed on phage were identified to specifically bind to 8H5 mAb. One of the five peptides, 123, was further displayed on the virus-like particle assembled from aa 1-149 fragment of HBcAg. The chimeric particle HBc-T123 conserved the specific binding to 8H5 mAb, and competed with H5N1 viruses for 8H5 mAb. The antiserum induced by HBc-T123 intensively stained on SF21 cells infected by recombinant baculovirus containing HA gene of YU22 virus, indicating the production of cross-reactive antibody to H5N1 HA.

[Adenovirus mediated double suicide gene under controlling by KDR promoter selectively kills vascular endothelial cells and colorectal tumor cells].

OBJECTIVE: To investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells. METHODS: 293 packaging cells were transfected with the plasmids of pAdEasy- KDR- CDglyTK and pAdEasy- CMV- CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT- PCR. After treatment with different concentrations of 5- FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined. RESULTS: The titers of these two purified Ads were 2.0 x 10(12 ) pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT- PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad- CMV- CDglyTK and the cells infected by Ad- KDR- CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows: (1) All cells infected with Ad- CMV- CDglyTK and some cells of ECV304 and SW620 infected with Ad- KDR- CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them (P > 0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs (P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene (P< 0.001). (3) The system had considerable bystander effect. CONCLUSION: The double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR- expressing endothelial cells and colorectal tumor cells.

[Construction of recombinant adenoviruses encoding TK suicide gene driven by VEGF promoter using efficient AdEasier-1 system].

BACKGROUND & OBJECTIVE: One of the bottlenecks of suicide gene therapy is the low specific expression of suicide genes in tumor cells. Using tumor specific promoter to modulate suicide genes resulting in high specific expression of genes in tumor cells has became a hot research topic of tumor suicide gene therapy. It has showed that vascular endothelial growth factor (VEGF) over-expresses in almost all solid tumors, but not in normal tissues, which is highly relevant to the up-regulation of VEGF promoter (VEGFP) activity. This study was to use the simplified and efficient AdEasier-1 system to generate recombinant adenoviruses encoding TK gene driven by VEGFP, and determine whether VEGFP could increase the expression of TK suicide gene in tumor cells. METHODS: A fragment containing VEGFP was isolated from pEGFP-1-SV-VEGFP (Not I /Xho I), and cloned into the shuttle plasmid pAdTrack resulting in pAdTrack-VEGFP. TK gene by polyadenylation site was cut from pREP8-TK by HindIII,and XbaI, and subcloned into pAdTrack-VEGFP resulting in pAdtrack-VEGFP-TK, which was linearized by PmeI and transformed into AdEasier-1 Cells. Transformants were selected on LB agar plates containing 25 microg/mL kanamycin, and positive pAdEasy-VEGFP-TK was identified by electrophoretic analysis and enzymatic digestion, then digested with PacI, and transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-TK. Ad-VEGFP-TK was finally confirmed by polymerase chain reaction (PCR) procedure, and DNA sequence analysis. RESULTS: The pAdTrack-VEGFP-TK (about 12 kb)and pAdEasy-VEGFP-TK(larger than 33 kb), both selectable by kanamycin resistance, could be clearly identified by electrophoretic analysis. Digesting pAdEasy-VEGFP-TK with PacI resulted in 1 specific small fragment (about 3.0 kb), and 1 large fragment (larger than 33 kb). The pAdEasy-VEGFP-TK was successfully constructed at a frequency of 60% (6/10). After being packaged in 293 cells, and purified by CsCl banding, the recombinant adenovirus Ad-VEGFP-TK, whose titer was as high as 5.6x1012 viral particle/ml, were produced, which were further proved to be correct by enzyme digestion, PCR analysis, and DNA sequence analysis. CONCLUSION: AdEasier-1 system is an efficient way to construct the recombinant adenoviruses encoding TK gene under control of VEGFP.