ABSTRACT
Objective To investigate the relationships and types between pulmonary subsolid nodules and bronchi and vessels,and their diagnostic values in differentiating subsolid nodules.Methods MSCT images of 40 subsolid nodules were retrospectively reviewed.The relationships between subsolid nodules and bronchi were divided into five types.Type Ⅰ:the bronchi were “cutoff”in the solid part of nodules.Type Ⅱ:the bronchi were distortion and extension in the solid parts of nodules.Type Ⅲ:the bronchi were distortion and extension in the GGO parts of nodules.Type Ⅳ:the bronchi were normal course in the GGO parts of nodules.Type Ⅴ:the bronchi were circumvent nodule lesions.The relationships between subsolid nodules and vessels were categorized into four types.Type Ⅰ:vessels passing by SSNs;Type Ⅱ:intact vessels passing through SSNs;Type Ⅲ:distorted,dilated or tortuous vessels seen within SSNs;Type Ⅳ:more complicated vessels other than described above.The relationship types were correlated to pathologic and/or clinical findings of subsolid nodules.Results Of 40 subsolid nodules,4 were preinvasive nodules,6 micro infiltrating adeocarcinomas and 30 invasive ones that were pathologically proven.Types Ⅰ,Ⅱ,Ⅲ and Ⅳ vascular relationships were observed in 0,8,21 and 11 subsolid nodules,respectively.Type Ⅲ relationship was the dominating one.All 11 subsolid nodules were invasive adenocarcinomas,when the relationship between subsolid nodules and vessle was type Ⅳ.There were 17 invasive adenocarcinomas,2 preinvasive ones,and 2 infiltrating ones when the relationship between subsolid nodules and vessel was type Ⅲ.Correlation analysis showed strong correlation between invasive adenocarcinoma and type Ⅲ and Ⅳ relationships.There was significantly statistical significance among 3 groups of relationships between subsolid nodule and vessels(χ2 =1 5.1 90,P =0.004).Types Ⅰ,Ⅱ,Ⅲ,Ⅳ and V brochi relationships were observed in 20,5,6,9 and 0 subsolid nodules,respectively.Type I relationship was the dominating relationship.There were 1 7 invasive adenocarcinomas, 1 preinvasive one,2 micro invasive ones when the relationship between subsolid nodules and bronchi were typeⅠ.There was significantly statistical significance among 3 groups of relationships between subsolid nodule and bronchi (χ2 =23.81 1,P =0.001 ).Conclusion Different subsolid nodules have different relationships with bronchi and vessels.Understanding and recognizing the characteristic subsolid nodule-bronchi and subsolid nodule-vessel relationships may help to identify which nodules are more likely to be malignant.
ABSTRACT
BACKGROUND: At present,enhanced green fluorescent protein(EGFP)is proved to be the best labeled molecule,with unique advantages,such as high fluorescence specificity,easy to be detected,and so on.Recombined retroviral vector EGFP-pLNCX2,which can stably express EGFP,can be construct using gene-engineering technique.Transfecting allogenic chondrocytes is indeed very useful to investigate the target gene expression and process of constructing tissue engineered cartilage in vivo.OBJECTIVE:To construct recombined retroviral vector EGFP-pLNCX2 which can stably express EGFP,and investigate optimal conditions for retrovirus transfection of chondrocytes.DESIGN:Randomized controlled observation.SETTING:Shanghai Jiao Tong University.MATERIALS:Thirty New Zealand rabbits of either gender,1 week of age,were purchased from Experimental Animal Center of Chinese Academy of Sciences.Amphotropic retrovirus package cell line PT67 pLNCX2 and pEGEP-C1 were purchased from Clontech Company;NIH 3T3 cell line was purchased from ATCC Company;DH5a Bacterium coli was preserved by the laboratory of Shanghai Jiao Tong University;Retroviral vector pLNCX2 was purchased from Clontech Company;pEGFP-C1 plasmid with EGFP were donated by professor Cong Xiao-qian from Chinese Academy of Sciences.METHODS:This experiment was carried out in the Shanghai Jiao Tong University in August 2005.Chondrocytes of New Zealand rabbits were isolated and cultured.The recombinant retroviral vector EGFP-pLNCX2,which can stably express EGFP,was constructed to transfect cultured chondrocytes from New Zealand rabbits by using gene engineering technique.Transfection results were observed under fluorescence microscope.Altogether 6×105 chondrocytes incubated in 10 cm-diameter flat plate were used to transfect retrovirus-EGFP immediately and at 12,24 and 48 hours after inoculation.One week later,EGFP expression efficiency was measured with flow cytometer,and best occasion for retrovirus transfecting primary chondrocytes.Enzyme-digested chondrocytes were inoculated for 24 hours to transfect retrovirus.After 250 mg/L G418 was added,chondrocytes were screened on the 2nd,3rd,4th,5th and 6th days separately.After phosphate buffer solution(PBS)washings,the transfection efficiency of chondrocytes was detected by flow cytometer,and the best occasion for G418 screening was observed.MAIN OUTCOME MEASURES:①EGFP-pLNCX2 transfection efficiency.②The best occasions for retrovirus transfecting primary chondrocytes and G418 screening.RESULTS: ①Retroviral vector EGFP-pLNCX2 transfected primary chondrocytes of rabbits,and high expression of transfected chondrocytes could be obtained through preliminary screening of G418.After being screened and expressing EGFP,chondrocytes kept normal morphology,with pseudopod adhering to the wall and matrix secreting vigorously.②The best occasion for retrovirus transfecting primary chondrocytes was at 24 hours after cell inoculation.The transfection efficiency determined with flow cytometer was 19.14% on the 5th day.The best occasion for G418 screening was on the 5th day after culture.Transfection efficiency of G418 screening was 55.75% on the 7th day.CONCLUSION:Recombinant retroviral vector EGFP-pLNCX2 can effectively transfect chondrocytes.The best occasion for retrovirus transfecting primary chondrocytes is at 24 hours after inoculation, and the best occasion for G418 screening is on the 5th day after culture.
ABSTRACT
BACKGROUND: The transplantation of allogeneic cartilage has local immunological rejection, and it is necessary to further reduce the rejection to promote its application in clinic, thus it is significant to perform a series of experiments to induce local immune privilege.OBJECTIVE: To observe the in vivo growth of tissue engineered allogeneic cartilage reconstructed by chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL.DESIGN: A randomized controlled observation.SETTING: Shanghai Jiao Tong University.MATERIALS: Thirty-six allogeneic New Zealand rabbits as recipients and 45 1-week-old chinchillas as donors, either sex,were purchased by the experimental animal center of Chinese Academy of Sciences. Amphotropic recombinant retrovirus coated cell line PT67 was purchased from Clontech Company; Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), G418 and Polybrene were bought from GIBCO BRL.METHODS: The experiment was carried out in original Shanghai Second Medical University from January 2000 to July 2005. The New Zealand rabbits were randomly divided into three groups: FasL-transfected group (n =12), untransfected group (n =12) and blank control group (n =12). The rabbit allogeneic cartilages were constructed by the compound of pLNCX2-FasL transfected chondrocytes and tissue engineered material of pluronic F-127. ① Gross observation and mass changes of the grafts: Corresponding materials were infused subcutaneously, the grafts were removed at 1, 2 and 3 months after transplantation for gross observation and the mass changes. ② Staining observation: The grafts were removed at 1, 2 and 3 months after transplantation, then prepared into sections, and observed by hematoxylin and eosin (HE), Safranin'O and Masson's trichrome stainings. ③ Antibody detection: Blood samples (1 mL) were collected at 1 and 2 months after transplantation, the chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, and separated by electrophoresis in agarose medium, then acted with serum of recipient to observe whether corresponding antibody generated. ④ Complement dependent cytotoxicity (CDC) test: The chondrocytes of chinchillas were prepared into cell suspension (2×109/L), and then seeded into 96-well plate, attached grew for 24 hours, then recipient serum was added for the CDC test, and the percentage of apoptotic cells was counted under microscope.MAIN OUTCOME MEASURES: ① Gross observation and mass changes of the grafts;② Histological changes; ③ Results of the antibody detection; ④ Percentage of apoptotic cells.RESULTS: All the 81 rabbits were involved in the analysis of results. ① Gross observation and mass changes of the grafts: Two weeks after inoculation, there were obvious nod formations at the inoculated sites, but no nod formed in the blank control group. The new cartilage tissues became smaller gradually and completely disappeared at 4 months in the untransfected group, whereas those in the FasL-transfected group became smaller, but still existed after 7 months. The masses of grafts in the FasL-transfected group were higher than those in the untransfected group (P ＜ 0.05). ②Histological observation: Plenty of lymphocytic infiltrations around cartilage tissue could be observed in the untransfected group, and obviously decreased in the FasL-transfected group. No lymphocyte was observed inside the chondrocytes.Masson's trichrome staining was performed, and it was observed under light microscope that the small white parts in the middle were immature chondrocytes, and there were green collagen around most of the mature chondrocytes. Safranin O staining showed strong positive reaction, suggested that there were rich glycosaminoglycan in matrix. ③ Antibody detection: The chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, then acted with serum of recipient, and the results showed that no corresponding antibody generated. ④ Percentage of apoptotic cells: The percentages of serum CDC apoptotic cells in the FasL can ransfected group, untransfected group and blank control group were 5%, 6% and 1%, which were all negative.CONCLUSION: Rabbit allogeneic chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL can reconstruct tissue engineered cartilage, and can postpone the degeneration by 3 months.