BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are easily isolated and amplified, and facilitate the exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are ideal therapeutic cells and target cells in the gene therapy.OBJECTIVE: To investigate liposome-mediated cytosine deaminase (CD) gene transfecting rabbit BMSCs and its gene expression. DESIGN: A single sample observation. SETTING: Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University.MATERIALS: This study was performed at in the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were included in this study. METHODS: The CD gene was obtained from E.coli JM109 DNA by polymerase chain reaction (PCR). The fragment was cloned into pMD19-T vector. After restriction enzyme BamHI/XhoI digestion analysis and DNA sequence analysis, pIRES2-AcGFP1-CD eukaryotic expression plasmid was constructed. Meanwhile, BMSCs were harvested, cultured and identified. After enzyme digestion of eukaryotic expression plasmid, the rabbit BMSCs were transfected by Lipofectamine 2000-mediated method. Twenty-four hours after transfection, expression of green fluorescent protein was observed under an inverted fluorescent microscope. MAIN OUTCOME MEASURES: Construction of eukaryotic expression plasmid and identification of CD gene-transferred BMSCs. RESULTS: CD gene was cloned and connected to eukaryotic expression plasmid with green fluorescence. Twenty-four hours after transfecting rabbit BMSCs, it was found under an inverted microscope that under the excitation of 488 nm blue light, green fluorescence appeared in the pIRES2-AcGFP1-CD and pIRES2-AcGFP1 empty-plasmid transfected BMSCs, but not in the non-transfected ones. It indicates that CD gene successfully transferred BMSCs. CONCLUSION: BMSCs are ideal vectors in the CD gene therapy.

The multipotent differentiation and immunosuppression capability of mesenchymal stem cells (MSCs) make it attractive source for stem cell therapy to treat serious diseases, including neural system diseases and immune disorders. For large scale clinical applications, MSCs have to be expanded to produce sufficient quantity for multiple treatments. While conventional passaging is not appropriate for such a task, bioreactor can be used to expand MSCs more efficiently. Yet the efficacy and biosafety of expanded MSCs must be properly assessed before the expanded MSCs can be implanted. This review presented state-of-the-art in expanding MSCs focusing on the progress on the assessment of the efficacy and biosafety of in vitro expanded MSCs. Current obstacles were discussed and future research directions were outlined.
Animals ; Bioreactors ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Colony-Forming Units Assay ; Humans ; Mesenchymal Stem Cell Transplantation ; adverse effects ; Mesenchymal Stromal Cells ; cytology

Objective:To investigate the expression of coxsackievirus-adenovirus receptor (CAR) in thymic carcinoma and the relationship between CAR and the antitumor activity of oncolytic adenovirus H101.Methods:The expression of CAR in thymic carcinoma tissues and cells were detected by RT-qPCR and Western blot. H101 expression and virus titers in Bcap-37, MP59 and T1889 cells after infection were detected by RT-qPCR and 50% tissue culture infectious dose (TCID 50). The proliferation activity and apoptosis rates of T1889 cells infected with H101 at different multiplicity of infection (MOI) were detected by CCK-8 and flow cytometry. CAR expression in T1889 cells treated with different concentrations of trichostatin A (TSA), a histone deacetylase inhibitor, was detected. H101 expression and virus titers in the TSA-treated and H101-infected cells were detected. Cell activity was detected by CCK-8. The phosphorylation levels of MARK and ERK1/2 and the expression of CAR at protein level in TSA-treated or TSA+ TBHQ (ERK activator) treated cells were detected. Results:CAR expression at both mRNA and protein levels were significantly lower in thymic carcinoma tissues than in adjacent normal tissues ( P<0.01), and lower in MP59 and T1889 cells than in thymic epithelial cells (TEC) and Bcap-37 cells ( P<0.01). H101 expression in MP59 and T1889 cells and the titers of H101 in culture supernatants were significantly lower than those in Bcap-37 cells ( P<0.01). Compared with Bcap-37 cell, the activity of MP59 and T1889 cells was significantly increased and the apoptosis rates were significantly decreased 48 h after H101 infection ( P<0.01). The expression of CAR at both mRNA and protein levels in T1889 cells treated with different concentrations of TSA increased in a dose-dependent manner. When T1889 cells were treated with 0.25 μmol/L of TSA, the expression of H101 at mRNA level and H101 titers were significantly increased ( P<0.05); the phosphorylation levels of MAPK and ERK1/2 proteins were continuously decreased; the expression of CAR was continuously increased. Compared with the TSA treatment group, the expression of CAR at protein level in the TSA+ TBHQ treatment group decreased significantly ( P<0.01), and the p-ERK1/2/ERK1/2 ratio increased significantly ( P<0.01). Conclusions:TSA could up-regulate CAR expression in thymic carcinoma by inhibiting the MARK/ERK1/2 pathway, thereby enhancing the antitumor activity of H101.

Objective:To explore the clinic effects of the head of second metatarsal bone and flap for bone and skin defect at lateral malleolus in children.Methods:Between July 2009 and May 2018, 4 children with ankle bone and skin and soft tissue defects in the Jizhong Energy Xingtai Mig General Hospital were selected, including 3 boys and 1 girl aged 4-13 years old. All cases were lateral malleolus defect with adjacent skin defect, the range of skin defect was 2.0 cm×2.0 cm -4.0 cm×5.0 cm, and the range of bone defect was 1.0-3.0 cm. The area of the flap was 2.5 cm×2.5 cm-4.5 cm × 5.5 cm, and the length of the second metatarsal head was 1.0-3.0 cm. The donor site was closed directly. After the operation, X-rays was performed to evaluate whether the ankle space and lateral malleolus longitudinal development was synchronized with the uninjured limb. Ankle joint function recovery was evaluated according to the American Orthopaedic Foot and Ankle Society (AOFAS). The outcomes of donor foot range of motion, weight-bearing, and walking were observed.Results:All the composite tissue flap survived with good blood circulation, and all the donor sites healed well. All the child patients were followed up for 0.5-2.0 years. The color and elasticity of the flaps were good, without cicatricial contracture. The patients had no inversion of ankle joint, with satisfying loading and walking function. Imaging evaluation showed that the space between ankle points and the longitudinal development of lateral malleolus were roughly synchronous with the healthy side. Two cases were graded as excellent and 2 as good according to the standard of the AOFAS.The donor's foot had normal flexion and extension function, without instability.Conclusions:The head of second metatarsal bone and flap can repair the epiphysis and soft tissue defect of lateral malleolus in children at one stage, and the reconstructed lateral malleolus can develop with the growth of children. It is a satisfactory method of reconstructing the traumatic defect of lateral malleolus in children.

Objective:To explore the safety and short-term and long-term efficacy of robot-assisted radical esophageal cancer surgery.Methods:A prospective randomized controlled trial was conducted. Patients who were preoperatively diagnosed as stage 0-IIIB esophageal squamous cell carcinoma and suitable for minimally invasive surgery in our hospital from January 1, 2014 to June 30, 2018 were prospectively enrolled. Those of age ≥75 years having received preoperative neoadjuvant therapy, contradicted to anesthesia or operation due to severe complications, with history of thoracotomy or laparotomy, with concurrent malignant tumors, without complete informations or refusing to participate in this study were excluded. Participants were randomly divided into the thoracoscopy-laparoscopy group and the robot group using a random number table in ratio of 1:1. Preoperative clinicopathological data, surgical data and postoperative outcomes were recorded. The patients were followed up mainly by telephone. Follow-up endpoint was recurrence of esophageal cancer and death. Kaplan-Meier method was used to estimate survival rate. The survival difference between the two groups was analyzed using the log-rank test.Results:According to above criteria, a total of 192 esophageal cancer patients were enrolled finally, including 144 males and 48 females with mean age of (61.9±8.6) years. The robot group had 94 cases, including 72 males and 22 females with mean age of (61.3±8.2) years, and the thoracoscopy-laparoscopy group had 98 cases, including 72 males and 26 females with mean age of (62.4±9.1) years. There were no significant differences in baseline data between the two groups (all P>0.05). Operation was abandoned in one case in each group due to extensive pleural cavity metastasis and one case in each group was converted to thoracotomy. The success rate of operation was 97.9% (92/94) in the robot group and 98.0% (96/98) in the thoracoscopy-laparoscopy group (χ 2=0.002, P=0.996). The number of lymph nodes dissected in the robot group was significantly higher than that in the thoracoscopy-laparoscopy group (29.2±12.5 vs. 22.8±13.3, t=3.433, P=0.001), while there were no significant differences in operative time, intraoperative blood loss, R0 resection rate, postoperative 30-day mortality, postoperative hospital stay, ICU stay, time to withdrawal of chest drainage tube, ICU readmission, and postoperative morbidity of complications between the two groups (all P>0.05). The median follow-up time was 21 (3 to 57) months. During the follow-up, 3 cases and 4 cases were lost, and 2 cases and 3 cases died of other diseases in the robot group and in the thoracoscopy-laparoscopy group respectively. Recurrence occurred in 39 cases during follow-up, including 14 recurrences in the robotic group with 1- and 3-year recurrence-free survival rates of 92.4% and 87.6% respectively and the median recurrence time of 15 (9 to 42) months. There were 25 recurrences in the thoracoscopy-laparoscopy group with 1- and 3-year recurrence-free survival rates of 81.7% and 67.9% respectively and the median recurrence time of 9 (3 to 42) months. There was significant difference in recurrence-free survival between the two groups (χ 2=4.193, P=0.041). Conclusions:The robotic surgical system has good oncology effect and surgical safety in the radical operation of esophageal cancer, which deserves further research and promotion.

Objective:To explore the safety and short-term and long-term efficacy of robot-assisted radical esophageal cancer surgery.Methods:A prospective randomized controlled trial was conducted. Patients who were preoperatively diagnosed as stage 0-IIIB esophageal squamous cell carcinoma and suitable for minimally invasive surgery in our hospital from January 1, 2014 to June 30, 2018 were prospectively enrolled. Those of age ≥75 years having received preoperative neoadjuvant therapy, contradicted to anesthesia or operation due to severe complications, with history of thoracotomy or laparotomy, with concurrent malignant tumors, without complete informations or refusing to participate in this study were excluded. Participants were randomly divided into the thoracoscopy-laparoscopy group and the robot group using a random number table in ratio of 1:1. Preoperative clinicopathological data, surgical data and postoperative outcomes were recorded. The patients were followed up mainly by telephone. Follow-up endpoint was recurrence of esophageal cancer and death. Kaplan-Meier method was used to estimate survival rate. The survival difference between the two groups was analyzed using the log-rank test.Results:According to above criteria, a total of 192 esophageal cancer patients were enrolled finally, including 144 males and 48 females with mean age of (61.9±8.6) years. The robot group had 94 cases, including 72 males and 22 females with mean age of (61.3±8.2) years, and the thoracoscopy-laparoscopy group had 98 cases, including 72 males and 26 females with mean age of (62.4±9.1) years. There were no significant differences in baseline data between the two groups (all P>0.05). Operation was abandoned in one case in each group due to extensive pleural cavity metastasis and one case in each group was converted to thoracotomy. The success rate of operation was 97.9% (92/94) in the robot group and 98.0% (96/98) in the thoracoscopy-laparoscopy group (χ 2=0.002, P=0.996). The number of lymph nodes dissected in the robot group was significantly higher than that in the thoracoscopy-laparoscopy group (29.2±12.5 vs. 22.8±13.3, t=3.433, P=0.001), while there were no significant differences in operative time, intraoperative blood loss, R0 resection rate, postoperative 30-day mortality, postoperative hospital stay, ICU stay, time to withdrawal of chest drainage tube, ICU readmission, and postoperative morbidity of complications between the two groups (all P>0.05). The median follow-up time was 21 (3 to 57) months. During the follow-up, 3 cases and 4 cases were lost, and 2 cases and 3 cases died of other diseases in the robot group and in the thoracoscopy-laparoscopy group respectively. Recurrence occurred in 39 cases during follow-up, including 14 recurrences in the robotic group with 1- and 3-year recurrence-free survival rates of 92.4% and 87.6% respectively and the median recurrence time of 15 (9 to 42) months. There were 25 recurrences in the thoracoscopy-laparoscopy group with 1- and 3-year recurrence-free survival rates of 81.7% and 67.9% respectively and the median recurrence time of 9 (3 to 42) months. There was significant difference in recurrence-free survival between the two groups (χ 2=4.193, P=0.041). Conclusions:The robotic surgical system has good oncology effect and surgical safety in the radical operation of esophageal cancer, which deserves further research and promotion.

Objective:To explore the clinic effects of the head of second metatarsal bone and flap for bone and skin defect at lateral malleolus in children.Methods:Between July 2009 and May 2018, 4 children with ankle bone and skin and soft tissue defects in the Jizhong Energy Xingtai Mig General Hospital were selected, including 3 boys and 1 girl aged 4-13 years old. All cases were lateral malleolus defect with adjacent skin defect, the range of skin defect was 2.0 cm×2.0 cm -4.0 cm×5.0 cm, and the range of bone defect was 1.0-3.0 cm. The area of the flap was 2.5 cm×2.5 cm-4.5 cm × 5.5 cm, and the length of the second metatarsal head was 1.0-3.0 cm. The donor site was closed directly. After the operation, X-rays was performed to evaluate whether the ankle space and lateral malleolus longitudinal development was synchronized with the uninjured limb. Ankle joint function recovery was evaluated according to the American Orthopaedic Foot and Ankle Society (AOFAS). The outcomes of donor foot range of motion, weight-bearing, and walking were observed.Results:All the composite tissue flap survived with good blood circulation, and all the donor sites healed well. All the child patients were followed up for 0.5-2.0 years. The color and elasticity of the flaps were good, without cicatricial contracture. The patients had no inversion of ankle joint, with satisfying loading and walking function. Imaging evaluation showed that the space between ankle points and the longitudinal development of lateral malleolus were roughly synchronous with the healthy side. Two cases were graded as excellent and 2 as good according to the standard of the AOFAS.The donor's foot had normal flexion and extension function, without instability.Conclusions:The head of second metatarsal bone and flap can repair the epiphysis and soft tissue defect of lateral malleolus in children at one stage, and the reconstructed lateral malleolus can develop with the growth of children. It is a satisfactory method of reconstructing the traumatic defect of lateral malleolus in children.

Objective:To investigate the clinical effects of retrograde anterolateral thigh perforator flaps assisted with computed tomography angiography (CTA) in repairing skin and soft tissue defects around the knee or in proximal lower leg.Methods:A retrospective cohort study was conducted. From May 2015 to October 2019, 17 patients with skin and soft tissue defects around the knee or in proximal lower leg were admitted to the Department of Orthopedics of Jizhong Energy Xingtai Mig General Hospital, including 12 males and 5 females, aged 16-65 years, with an average age of 35 years. The areas of skin and soft tissue defects after debridement ranged from 6.0 cm×3.0 cm to 15.0 cm×9.0 cm. The retrograde anterolateral thigh perforator flaps were designed according to the origin and distribution of the perforating branches in flaps and the length of the vascular pedicle examined with CTA and the condition of the wound to repair the wounds. The areas of resected flaps ranged from 6.5 cm×3.5 cm to 15.5 cm×9.5 cm. The wounds in donor sites of flaps were sutured directly or covered with medium-thickness skin grafts from healthy upper leg. The sources of the perforating branches in flaps were recorded. The lateral circumflex femoral artery, its branches, and the relative length of the vascular pedicle were compared between preoperative CTA detection and intraoperative observation. The survivals of the flaps were observed. At the last follow-up, the effects of flaps in repairing wounds were evaluated according to evaluation standard of efficacy satisfaction; the motion ranges of flexion and extension of the knee joint were measured, and the knee joint function was evaluated according to the Hohl knee joint function evaluation standard; the sensory function in the flap area was evaluated according to the sensory function evaluation standard formulated by the British Medical Research Council; the wound healing and the occurrence of complication affecting motor function of limb of flap donor sites was observed. Data were statistically analyzed with paired sample t test. Results:The perforating branches in flaps originated from descending branches, oblique branches, and rectus femoris branches of lateral circumflex femoral artery in 7, 6, and 4 patients, respectively. The flaps with blood supply from descending branches, oblique branches, and rectus femoris branches of lateral circumflex femoral artery were type 1, 2, and 3 retrograde anterolateral thigh perforator flaps, respectively. The preoperative CTA examination of lateral circumflex femoral artery and its branches were consistent with those observed during operation. The relative lengths of vascular pedicles of type 1, 2, and 3 retrograde anterolateral thigh perforator flaps calculated after CTA examination were 0.32±0.13, 0.56±0.07, and 0.56±0.15, which were close to 0.35±0.12, 0.52±0.10, and 0.53±0.12 measured and calculated during operation, respectively ( t=0.45, 0.80, 0.31, P>0.05). All flaps survived in 17 cases without vascular crisis. At the last follow-up, 16 patients were satisfied with effects of flaps in wound repair, with 1 patient feeling average about the effect; the flexion range of knee joint was 100-120°, and the extension range of knee joint was -2-0°; knee joint function was evaluated as excellent in 9 cases, good in 7 cases, and poor in 1 case; the sensory function of the flap area reached S 4 level in 2 cases, S 3 level in 8 cases, and S 2 level in 7 cases; the wounds in flap donor sites healed well; there was no adverse effect in motor function of limbs. Conclusions:Retrograde anterolateral thigh perforator flap is an effective method for repairing skin and soft tissue defects around the knee or in proximal lower leg. Preoperative CTA examination can fully show the anatomical characteristics of the branches of the lateral circumflex femoral artery and the perforating vessels of each branch, which can guide preoperative flap design and operation, thus shortening operation time and improving flap survival rate, with good clinical effects.

Pharmacy active consultation refers to the spontaneous activity that clinical pharmacists take the initiative to go to clinical departments to help doctors solve problems related to drug use in clinical practice ，put forward drug treatment suggestions or provide pharmaceutical services ，and form medical documents . The difference between pharmacy active consultation and pharmacy consultation is that the latter is generally proposed by the clinician ，who sends a consultation invitation to the pharmacy department in the hospital information system ，and the clinical pharmacist will go to the consultation after receiving it ，while the former is a pharmaceutical service mode that the clinical pharmacist takes the initiative to carry out in the clinical department . On the basis of routine pharmacy active consultation ，clinical pharmacists in our hospital also further carried out a special active consultation mode （including prompt special active consultation for patients with multidrug resistance bacteria positive ，active monitoring and intervention for patients with drug -induced liver injury ），and patient pharmaceutical supervision in the form of return visit of pharmacy active consultation . Pharmacy active consultation and its special active consultation possess the characteristics of initiative ， early and extensive coverage ，as a supplement to resident clinical pharmacy services . Pharmacy active consultation could help the pharmacy department to improve service efficiency ，provide a new perspective for medical institutions to carry out efficient pharmaceutical services ，and supply new ideas for the reform of pharmaceutical services in China .