Purpose:To construct replication-defective adenovirus which was recombinated with Egr-1 promoter and to Smad7, and to study whether it can express exogenous Smad7 protein in cytoplasm.Methods:Based on Adeno-X~(TM) expression system, the CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-X~(TM) genome, which was transformed into E.coli to get recombinant Adeno-X~(TM) plasmid DNA. The recombinant adenovirus was made and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. Then the Smad7’s location in cells was determined by immunocytochemistry.Results:Identified by restriction endonuclease analysis and PCR, recombinant adenovirus with Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0?10~(11) TCID50/ml. Both endogenous and exogenous Smad7 protein was found to be located in cytoplasm of fibroblast cells.Conclusions:With Adeno-X~(TM) expression system, utilizing the techniques of molecular cloning, recombinant adenoviral vector could be quickly and efficiently constructed which could be packaged into replication-defective Adenovirus and amplified in HEK293 cells. The recombinant adenovirus could express exogenous Smad7 protein in fibroblast cells.[

Background and purpose:Radiotherapy (RT) is one of the most important therapeutic tools for esophageal cancer. Because tumors are heterogeneous, including for18F-FDG uptake and, most likely, for radioresistance, selective boosting of high FDG uptake zones within the tumor has been suggested. Therefore, it is critical to know whether the location of these high FDG uptake patterns within the tumor remains stable during RT.Methods:Twenty-two patients with esophageal squamous cell carcinoma treated with concurrent chemo-radiation underwent repeated18F-FDG PET-CT scans before RT and after 20 fractions of RT. On all scans, the high and low FDG uptake regions were auto-delineated using several standard uptake value (SUV) thresholds, varying from 40% to 70% of SUVmax on the pretreatment scan [gross tumor volume (GTV)40%pre, GTV50%pre, GTV60%pre, GTV70%pre] and from 70% to 90% of SUVmax on the dur-treatment scan (GTV70%dur, GTV80%dur, GTV90%dur) and ifxed thresholds of 2.5 and 5 (GTV2.5pre, GTV5pre). The volumes and overlap fractions (OF) of these delineations were calculated to demonstrate the stability of the high FDG uptake regions during RT.Results:The high uptake regions within the tumor during RT largely corresponded (OF>70%) with the 50% SUVmax high FDG uptake area (GTV50%pre) of the pretreatment scan. The hotspot within the residual area (GTV90%dur) was completely within the GTV and pre-radiotherapy high uptake regions (OF=100%). Although the location of the high FDG uptake patterns within the tumor during RT remained stable, the delineated volumes varied markedly.Conclusion:The location of the high FDG uptake areas within the tumor remained stable during RT. This knowledge may enable selective boosting of high FDG uptake areas within the tumor.

Objective To investigate the time-effect relationship and dose-effect relationship of the expression of adenovirus mediating Smad 7 gene regulated by irradiation via Egr-1 promoter in the lung of C57BL mice. Methods Recombinant adenovirus (AD.Egr-Smad 7) was made up through replication-defective adenovirus enclosed radio-inducible elements from the Egr-1 gene promoter and cDNA encoding Smad 7. Mice infected by intratracheal instillation with AD.Egr-Smad 7 were irradiated to their whole lungs after 24 hours. 324 mice were randomly divided into 6 groups, and the lungs were harvested at different time points following irradiation with single dose of 8?Gy to observe the time-effect relationship of Smad7 gene expression with different time intervals. 108 mice randomly divided into 3 groups were irradiated respectively by different single irradiation dose and the lungs were harvested 5 hours after radiation to evaluated the dose-effect relationship of Smad 7 gene expression with different radiation doses. The expression of exogenous Smad 7 was detected by Western blot analysis. Results The expression of adenovirus mediating Smad 7 gene regulated by Egr-1 promoter could be induced by radiation markedly as compared with the control groups(P

Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.

Objective Objective To study the effect of adenovirus mediating Smad 7 gene regulated by radiation via Egr-1 on the primary tumor and lung metastasis in C57BL mice implanted with Lewis lung cancer.Methods The radio-inducible elements from the Egr-1 gene promoter were inserted upstream to a cDNA encoding Smad7 and integrated into a replication-defective adenovirus to generate recombinant adenovirus(AD.Egr-Smad 7).270 mice implanted with Lewis lung cancer in the hind legs were used and the experiment was started when the transplanted tumor diameter reached 0.8 to 1.0cm.Then three investigations were undertaken,each demanding 90 mice implanted with Lewis lung cancer respectively.To each group,90 mice models were randomized into 3 groups: the normal control group;the NS control group;and the implanted AD.Egr-Smad 7 group.Every 6 mice in each group were irradiated by different single dose to study the following: 1.The maximal and minimal diameters of the tumor were recorded to observe the tumor growth tendency,the tumor growth delay and the mice survival time,2.The incidence of lung metastasis two weeks after the radiation was recorded.3.The incidence of lung metastasis when the tumor volume was four times as large as that at the beginning of radiation was recorded.Results The adenovirus mediating Smad 7 gene expression regulated by irradiation via Egr-1 in C57BL mice implanted with Lewis lung cancer was able to inhibit the progression of the primary tumor and prolong the survival of the mice significantly as compared with the control group(P0.05).Conclusions The gene expression of AD.Egr-Smad 7 regulated by radiation is not risky in promoting the local progression and distant metastasis of Lewis lung cancer in mice.On the other hand,the gene expression of AD.Egr-Smad 7 regulated by radiation could inhibit the progression of the primary tumor and prolong the survival time of the mice significantly.It is safe,to some extent,of using AD.Egr-Smad 7 to block the signal transduction of TGF-?locally as a novel strategy for gene therapy aiming at the prevention of radiation-induced lung

Background and purpose: The prognosis of completely resected stage ⅢA(N2) non-small cell lung cancer (NSCLC) remains a significant concern. The 5-year overall survival (OS) rates range from 10% to 30%. This study aimed to analyze the patterns of first failure in completely resected stage ⅢA(N2) NSCLC and to assess the actuarial risk of developing metastasis at different sites and to guild standard clinical practice. Methods: Patients withⅢA(N2) NSCLC who had undergone radical surgery in our hospital from Jan. 2005 to Jul. 2012 were retrospectively reviewed. The progression-free survival (PFS), the OS, patterns of first failure, the actuarial risk were analyzed. The cumulative incidence of first failure was determined using the Kaplan-Meier analysis. Results: Among 357 patients who met the eligibility criteria with completely resected stage ⅢA(N2) NSCLC, 5-year OS was 36.9%. There were 284 (77.6%) patients experiencing disease failure: 61 with local failure, 197 with local and distant failures, and 26 patients with local recurrence as the first failure. Brain, bone and lung were the main sites of distant failure as the first failure, while brain was the most common site. There were 67 patients developing brain metastases (BM) as the first site of failure. The median time of local failure as the first site of failure was 13.6 months, and the time to develop distant recurrence was 15.1 months. 92.5% BM developed in 3 years after the complete resection. Conclusion: As the first failure, the rate of distant failure was much higher than that of local failure in completely resected stage ⅢA(N2) NSCLC. Brain was the most common site of distant failure as the first failure. These results can be helpful in guiding standard clinical practice and evaluating the outcome of comprehensive treatment.

Objective To study the effect of different dose fractionation on overall survival in patients with limited-stage small cell lung cancer (LS-SCLC). Methods LS-SCLC patients treated with radical combined chemotherapy and radiotherapy (RT) between January 2001 and Dec 2007 were analyzed retrospectively. According to the dose fractionation schemes, patients were divided into three groups:conventional fractionated RT (1. 8 -2.0 Gy,once daily), hyperfractionated RT (1.4 Gy, twice daily) and hypofractionated RT (2. 5 Gy,once daily). Overall survival, disease free survival and pattern of failures of the three groups were compared. A total of 177 patients were enrolled, including 63 patients in conventional fractionated RT group, 79 in hyperfractionated RT group and 35 in hypofractionated RT group. Results The overall follow-up rate was 96. 6%. The patient numbers with follow-up of more than 2 and 5 years were 153 and 92, respectively. The median survival time of the entire group was 22. 4 months, and the 2-and 5-year survival rates were 43.4% and 23. 5%, respectively. The 2-year survival rates for three groups were 31%, 46% and 59% (x2 =7.94,P=0.019), respectively. The 2-year disease free survival for three groups were 20%, 31% and 40% ( x2 = 4. 86, P = 0. 088 ), respectively. In the pairwise comparisons,patients in hypofractionated RT group have better survival than those in conventional fractionated RT group ( x2 = 7. 81, P = 0. 005 ), the effect of hyperfractionated RT group lies between the hypo-and the conventional fractionated RT groups, but no significant differences were detected ( x2 = 2. 31, P = 0. 128; x2 = 2. 95, P =0. 086). The mildest side effect was found in the hypofractionated RT group. No statistically significant differences were found in the patterns of first failure. Conclusion The hypofractionated RT scheme showed potential survival benefits for patients with LS-SCLC and should be considered in the setting of randomized clinical trials.

Objective To retrospectively design an intensity?modulated radiotherapy ( IMRT) plan with split field and fixed jaw techniques for peripheral lung cancer with mediastinal lymph node metastasis, to compare dosimetric characteristics between the IMRT plans with fixed jaw and dynamic jaw, and to study lung protection by the plan with split field and fixed jaw. Methods Treatment plans were collected from 12 patients with peripheral lung cancer and mediastinal lymph node metastasis who were treated with IMRT in our hospital. All plans used the dynamic jaw technique. The plans with split field and fixed jaw were designed based on the identical computed tomography images and planning target volume ( PTV) . Each jaw position in split field depended on each separate PTV. The prescription dose was 60 Gy in 30 fractions. 95%PTV was planned to receive 100% of the prescription dose. Dosimetric parameters of PTV, conformity index ( CI) , heterogeneity index ( HI) , number of monitor units ( MUs) , and dose?volume values of the lung and heart were obtained from dose?volume histogram. Comparison between the two plans was made by paired t test. Results Both plans met clinical requirements. There were no significant differences in D2 , D98 , CI, or HI of PTV between the two plans ( all P>005) . Compared with the dynamic jaw plan, the fixed jaw plan had MUs increased by 15%?20%( P=0010) . The V5 , V10 , V20 , V30 , and mean dose for the whole lungs were significantly lower in the fixed jaw plan than in the dynamic jaw plan ( P=0000, 0000, 0000, 0002,0000) . The V5 , V20 , and mean dose for the healthy lung were also significantly lower in the fixed jaw plan than in the dynamic jaw plan ( P=0000,0017,0000) . There were no significant differences in dose?volume values for the spinal cord or heart between the two plans ( all P>005) . Conclusions IMRT with split field and fixed jaw is recommended for patients with peripheral lung cancer and mediastinal lymph node metastasis. The therapy to a certain extent reduces low?dose volume for the lung and the incidence of radiation?induced pneumonitis.

Objective To comparative study the effect on colorectal cleansing of CT colonography with gulping down 10 mg bisacodyl before or 1 h after oral taking 2 liter polyethylene glycol .Methods Forty participants with informed consent were appor‐tioned to group A ,group B randomly ,20 cases in each group .On the day before CT colonography ,participants in group A oral took 20 mL of 40% W/V barium sulfate prior to 3 mealtime ,and 20 mL of 60% diatrizoate meglumine diluted in 250 mL of water after supper ,then gulped down 10 mg bisacodyl enteric‐coated tablets 1 hour before oral taking 2 liter polyethylene glycol electrolyte so‐lution .Participants in group B were the same as that in group A ,with the exception of gulping down 10 mg bisacodyl enteric‐coated tablets 1 hour after oral taking 2 liter polyethylene glycol electrolyte solution .Cleansing efficacy of stool and fluid ,and attenuation value of remainder fluid between the two groups were analyzed statistically .Results In group A ,score of cleansing efficacy of stool (1 .96 ± 0 .11) was lower than that in group B (2 .01 ± 0 .12) ,segments with good cleansing efficacy of stool (87/120 segments , 72 .50% ) was higher than that in group B (83/120 segments ,69 .17% ) ,the difference was not statistically significant (P>0 .05) .In group A ,score of cleansing efficacy of fluid (1 .50 ± 0 .06) was lower than that in group B (1 .53 ± 0 .06) ,segments with good cleansing efficacy of fluid(113/120 segments ,94 .17% ) was higher than that in group B (111/120 segments ,92 .50% ) ,the differ‐ence was not statistically significant (P>0 .05) .Attenuation value of remainder fluid [(729 ± 29)HU ] in group A was higher than that in group B[(653 ± 25)HU] ,the difference was statistically significant(P<0 .05) .Conclusion Gulping down 10 mg Bisacodyl before or after oral taking 2 liter polyethylene glycol has no effect on cleansing of stool and fluid ,with good cleansing efficacy .The former has better cleansing efficacy of fluid ,is beneficial to detecting polyps for CT colonography .

Objective To analyze radiation induced alterations of vitronectin and collagen expressions in fibroblasts at different times post-irradiation,so as to evaluate the potential to apply vitronectin as a biomarker of radiation-induced lung fibrosis.Methods The human fibroblast cells WI-38 and IMR-90 were irradiated with 137Cs γ-rays at doses of 0 (control),4,6,8,10 and 12 Gy,respectively.The cells and its supernatant were collected at 6,12,24,36,48 and 60 h post-irradiation.The expressions of vitronectin and collagen Ⅰ and Ⅲ were analyzed by Western blot,PCR and ELISA.Results After irradiation,the expressions of vitronectin and collagen Ⅰ and Ⅲ were positively correlated (r=0.40-0.79,P＜0.05) and were all significantly higher than that in control group (t =3.04-25.45,P ＜0.05) and reached the highest expression levels at 48 h after 8-10 Gy of irradiation (t =2.92-18.86,P ＜ 0.05).Analyses of Real-time PCR and ELISA assay showed that expressions of vitronectin mRNA and its protein level in the cell lysis were significantly increased by radiation (F =27.09-42.62,P ＜ 0.05).Conclusions The expressions of vitronectin in cellular supernatant and its mRNA may be a potential biomarker of radiation-induced fibrosis,and 48 h after 8 Gy irradiation may be an optimum condition of measurement.