[Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats].

Objective: To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats. Methods: The research was an experimental research. Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method, and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels (hereinafter referred to as hydrogels). Besides, the hydrogels loaded with mouse adipose-derived stem cells were prepared. Thirty male 12-week-old guinea pigs were divided into negative control group, positive control group, and hydrogel group according to the random number table, with 10 guinea pigs in each group. Ethanol, 4-aminobenzoic acid ethyl ester, or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact, and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact. Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells. After 3 d of culture, protein expressions of platelet-derived growth factor-D (PDGF-D), insulin-like growth factor-Ⅰ (IGF-Ⅰ), and transforming growth factor ß1 (TGF-ß1) were detected by Western blotting (n=3). Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back. The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied. Wound healing was observed at 0 (immediately), 2, 4, 8, and 10 d after injury, and the wound healing rate was calculated at 2, 4, 8, and 10 d after injury. Wound tissue samples at 10 d after injury were collected, the new tissue formation was observed by hematoxylin-eosin staining; the concentrations of interleukin-1α (IL-1α), IL-6, IL-4, and IL-10 were detected by enzyme-linked immunosorbent assay method; the expressions of CD16 and CD206 positive cells were observed by immunohistochemical staining and the percentages of positive cells were calculated. The sample numbers in animal experiment were all 8. Results: At 24 h after stimulated contact, no skin edema was observed in the three groups of guinea pigs, and only mild skin erythema was observed in 7 guinea pigs in positive control group. At 48 h after stimulated contact, skin erythema was observed in 8 guinea pigs and skin edema was observed in 4 guinea pigs in positive control group, while no obvious skin erythema or edema was observed in guinea pigs in the other two groups. After 3 d of culture, the protein expression levels of PDGF-D, IGF-I, and TGF-ß1 in adipose-derived stem cells in hydrogel group were significantly higher than those in normal control group (with t values of 12.91, 11.83, and 7.92, respectively, P<0.05). From 0 to 10 d after injury, the wound areas in both groups gradually decreased, and the wounds in hydrogel group were almost completely healed at 10 d after injury. At 4, 8, and 10 d after injury, the wound healing rates in hydrogel group were (38±4)%, (54±5)%, and (69±6)%, respectively, which were significantly higher than (21±6)%, (29±7)%, and (31±7)% in blank control group (with t values of 3.82, 3.97, and 4.05, respectively, Pvalues all <0.05). At 10 d after injury, compared with those in blank control group, the epidermis in wound in hydrogel group was more intact, and there were increases in hair follicles, blood vessels, and other skin appendages. At 10 d after injury, the concentrations of IL-1α and IL-6 in wound tissue in hydrogel group were significantly lower than those in blank control group (with tvalues of 8.21 and 7.99, respectively, P<0.05), while the concentrations of IL-4 and IL-10 were significantly higher than those in blank control group (with tvalues of 6.57 and 9.03, respectively, P<0.05). The percentage of CD16 positive cells in wound tissue in hydrogel group was significantly lower than that in blank control group (t=8.02, P<0.05), while the percentage of CD206 positive cells was significantly higher than that in blank control group (t=7.21, P<0.05). Conclusions: The hydrogel loaded with mouse adipose-derived stem cells is non-allergenic, can promote the secretion of growth factors in adipose-derived stem cells, promote the polarization of macrophages to M2 phenotype in wound tissue in rats with full-thickness skin defects, and alleviate inflammatory reaction, thereby promoting wound healing.

[Clinicopathological features and prognosis of 47 adults with Hirschsprung's disease and Hirschsprung's disease allied disorders].

Objective: To improve understanding and treatment of adult Hirschsprung's disease (HD) and Hirschsprung's disease allied disorders (HAD) by investigating the clinicopatho- logical features, diagnostic and treatment methods, and prognosis. Methods: This was a retrospective observational study. The study cohort comprised patients aged 18-65 years admitted to the Sixth Hospital of Sun Yat-sen University between January 2007 and December 2022 who were diagnosed with adult HD or HAD by postoperative pathological examination. Those with severe cardiovascular disease, diabetes mellitus, or cirrhosis of the liver were excluded, leaving 47 patients in the study cohort. Emergency open surgery was performed on patients with life-threatening manifestations, whereas those whose condition was stable received conservative treatment to stabilize them, following which they underwent a standard surgical procedure. Surgical procedures performed included the Duhamel procedure, Soave procedure, subtotal colonic resection, total colonic resection, and creation of a palliative stoma. Variables studied included clinicopathological characteristics, treatment modalities, postoperative complications, and long-term anal function. Complications were evaluated in accordance with the Clavien-Dindo criteria, and long-term anal function according to the 2005 Krickenbeck International Classification Criteria. Results: Of the 47 patients, 33 were men and 14 women, with a median age of 29 (18-51) years. HD was diagnosed in 41 (87.2%) patients and HAD in six (12.8%). The commonest initial symptom was dyspareunia (70.2%,33/47), followed by abdominal distension (57.4%, 27/47) and abdominal pain (44.7%,21/47). The detection rates of HD/HAD by barium enema + defecography, anorectal manometry, and preoperative rectal biopsy were 86.8% (33/38), 16/19, and 7/7, respectively. Three (6.4%) patients had discrepant preoperative clinical and postoperative pathological diagnoses. None of the three misdiagnosed patients had undergone preoperative rectal biopsy. Of the 47 study patients, three chose non-surgical treatment and 44 surgical treatment. All surgeries were successfully completed. Postoperative complications occurred in 19 patients (43.2%), including one death case who had undergone emergency surgery. The median duration of follow-up after surgery was 65 (12-180) months. Three patients in the surgical treatment group were lost to follow-up. Of the remaining 41 patients, 36, three, and two had excellent, good, and poor long-term anal function, respectively. The differences in outcomes between the surgical and non-surgical treatment groups (no patients, one, and two with excellent, good, and poor long-term anal function, respectively) (Z=-3.883, P=0.001) were statistically significant. Of the 44 patients who underwent surgical treatment, 41 underwent standard surgeries and three emergency surgeries because their conditions were life-threatening. The difference in complication rate between standard surgery and emergency surgery groups (39.0% [16/41] vs. 3/3, χ2=2.115, P=0.146) was not statistically significant. However, the rate of postoperative Grade III-V complications was lower in the standard surgery group (4.9% [2/41] vs. 2/3, Z=-2.668, P=0.008). Long-term anal function was significantly better in the standard surgery than emergency surgery group (94.7% [36/38] vs. 0/3, Z=-4.935, P=0.001). The 41 standard surgeries included 11 Duhamel's procedures, six Soave's procedures, 19 subtotal colonic resections, three total colonic resections, and two palliative colostomies. The incidence of postoperative complications was significantly superior in the Duhanmels procedures and palliative colostomies group(1/11 and 0/2, P=0.041). Of the 41 patients who underwent standard surgery, 23 underwent open surgery and 18 minimally invasive laparoscopic surgery. The incidence of postoperative Grade III-V complications and long-term anal function were significantly superior in the laparoscopic group than in the open group (all P<0.05). Conclusion: It is easy to misdiagnose adult HD and HAD, surgical treatment is safe and feasible, and its long-term efficacy is good.

[Analysis of perioperative efficacy and safety of cytoreductive surgery in the treatment of colorectal cancer peritoneal metastases].

Objective: To analyzed perioperative safety of cytoreductive surgery (CRS) for patients with colorectal cancer peritoneal metastasis (CRPM) and to construct a predictive model for serious advese events (SAE). Methods: A descriptive case-series study was conducted to retrospectively collect the clinicopathological data and treatment status (operation time, number of organ resection, number of peritoneal resection, and blood loss, etc.) of 100 patients with peritoneal metastases from colorectal cancer or appendix mucinous adenocarcinoma who underwent CRS at the Sixth Affiliated Hospital of Sun Yat-sen University from January 2019 to August 2021. There were 53 males and 47 females. The median age was 52.0 (39.0-61.8) years old. Fifty-two patients had synchronous peritoneal metastasis and 48 had metachronous peritoneal metastasis. Fifty-two patients received preoperative neoadjuvant therapy. Primary tumor was located in the left colon, the right colon and the rectum in 43, 28 and 14 cases, respectively. Fifteen patients had appendix mucinous adenocarcinoma. Measures of skewed distribution are expressed as M (range). Perioperative safety was analyzed, perioperative grade III or higher was defined as SAE. Risk factors associated with the occurrence of SAEs were analyzed using multivariate logistic regression. A nomogram was plotted by R software to predict SAE, the efficacy of which was evaluated using the area under the ROC curve (AUC) and correction curves. Results: The median peritoneal cancer index (PCI) score was 16 (1-39). Sixty-eight (68.0%) patients achieved complete tumor reduction (tumor reduction score: 0-1). Sixty-two patients were treated with intraperitoneal hyperthermic perfusion chemotherapy (HIPEC). Twenty-one (21.0%) patients developed 37 SAEs of grade III-IV, including 2 cases of ureteral injury, 6 cases of perioperative massive hemorrhage or anemia, 7 cases of digestive system, 15 cases of respiratory system, 4 cases of cardiovascular system, 1 case of skin incision dehiscence, and 2 cases of abdominal infection. No grade V SAE was found. Multivariate logistic regression analysis showed that CEA (OR: 8.980, 95%CI: 1.428-56.457, P=0.019), PCI score (OR: 7.924, 95%CI: 1.486-42.259, P=0.015), intraoperative albumin infusion (OR: 48.959, 95%CI: 2.115-1133.289, P=0.015) and total volume of infusion (OR: 24.729, 95%CI: 3.956-154.562, P=0.001) were independent risk factors for perioperative SAE in CRS (all P<0.05). Based on the result of multivariate regression models, a predictive nomogram was constructed. Internal verification showed that the AUC of the nomogram was 0.926 (95%CI: 0.872-0.980), indicating good prediction accuracy and consistency. Conclusions: CRS is a safe and effective method to treat CRPM. Strict screening of patients and perioperative fluid management are important guarantees for reducing the morbidity of SAE.

[Meta analysis of whether cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy can improve survival in patients with colorectal cancer peritoneal metastasis].

Objective: To explore whether the cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS+HIPEC) can improve the survival rate of colorectal cancer patients with peritoneal metastasis. Methods: The relevant studies were systematically retrieved from PubMed, Embase, Cochrane Library, CNKI, Wanfang, VIP database, and the study of French Elias' team on peritoneal metastasis was retrieved manually. Inclusion criteria: (1) The patients were colorectal cancer peritoneal metastasis. (2) There were CRS+HIPEC treatments (treatment group) and other treatments (control group). (3) Survival analysis data of treatment group and control group were available. (4) Types of studies were randomized controlled trials, cohort studies, or case-control studies. (5) The literature was in Chinese or English. Exclusion criteria: (1) studies without full-text; (2) studies without complete data. The literature screening and data extraction were carried out by two people independently, and the third person decided on the literature with differences. The extracted data included authors, year of publication, number of patients, time of enrollment, time of follow-up, studies design, treatment regimen, hazard ratio (HR) and 95% CI of treatment group and control groups. If the HR and 95% CI of the treatment group and control group were not provided in the literature, Engauge Digitizer 11.1 software was used to extract the time of follow-up and the survival rate at the corresponding time point from the survival curves of both groups, and the HR and 95% CI of both groups were calculated by combining the number of both groups. The quality of study was evaluated by Newcastle-Ottawa scale (NOS) or Cochrane collaboration's tool for assessing risk bias. STATA 15.1 software was used for statistical analysis. HR and 95% CI of both groups were pooled and analyzed. Inter-trial heterogeneity was assessed by Q test and I(2) statistics. When there was no significant heterogeneity (Q test: P≥0.10), fixed-effect model was used for pooled analysis. When significant heterogeneity existed (Q test: P<0.10), random effect model was used for pooled analysis, and subgroup analysis was used to find out the source of heterogeneity. Sensitivity analysis was used to evaluate the stability of the pooled results. Publication bias was assessed by Egger's test and Begg's test (P<0.05 indicated publication bias) and it is reflected by the visual symmetry of Begg's funnel plot on the natural logarithm of HR. Results: A total of 10 studies were enrolled in the meta-analysis, including 1 randomized controlled trial and 9 cohort studies. The risk of bias in 1 randomized controlled trial was uncertain, and 9 cohort studies were all higher than 7 points, indicating high quality literatures. There were 781 patients in treatment group receiving CRS+HIPEC and 2452 patients in control group receiving other treatment, including tumor cytoreductive surgery (CRS), palliative chemotherapy (PC) and intraperitoneal chemotherapy (IPC). The results of pooled analysis by random effect model showed that the OS rate in treatment group was significantly higher than that in control group (HR=0.43, 95% CI: 0.34-0.54), but the heterogeneity of the study was high (P=0.024, I(2)=52.9%). The subgroup analysis of different control treatments showed that the OS rate in treatment group was significantly higher than that in CRS control group (HR=0.63, 95% CI: 0.44-0.90), in PC control group (HR=0.37, 95% CI: 0.32-0.43), in CRS+ IPC control group (HR=0.60, 95% CI: 0.37-0.96), and the heterogeneity of each subgroup was low (CRS control group: P=0.255, I(2)=22.9%; PC control group: P=0.222, I(2)=29.9%; CRS+IPC control group: P=0.947, I(2)=0). Due to the low heterogeneity of subgroups, fixed-effect models were used to pool and analysis. The results of sensitivity analysis revealed that there was little difference between the pooled analysis results after each study was deleted, suggesting that the pooled analysis results were more reliable. Publication bias detection of each study showed Begg's test (P=0.088) >0.05 and Egger's test (P=0.138)>0.05. According to the Begg's funnel plot, the scatter point distribution was basically symmetric, indicating that there was no publication bias in the included study. Conclusion: CRS+HIPEC can improve the OS of patients with colorectal cancer peritoneal metastasis.

CD34 positive cells seeded on small caliber man-made vascular grafts exhibit increased antithrombogenic property compared with unfractioned mononuclear cells.

AIM: The aim of this study was to compare the therapeutic potential of purified CD34+ cells with that of unfractioned mononuclear cells (MNCs) for the antithrombogenic property after seeding on the small caliber man-made grafts. METHODS: Unfractioned MNCs and CD34+ cells were isolated from canine bone marrow. Differentiation of CD34 cells and unfractioned MNCs into endothelial cells were examined by CD31and vWF immunohistochemistry or immunofluorescence stain, endothelial cell function were evaluated via low-density lipoprotein (ac-LDL) - DiL incorpration and in vitro angiogenesis assay. Platelet adhesion assay was performed to determine antiplatelet adhesion property of the cells in vitro. Equal number of both cells were seeded onto the luminal surface of small caliber man-made grafts and implanted to replace a segment of common carotid artery. At different time points (24 h, 72 h, and 1 week) the grafts were retrieved. HE staining and SEM exam were performed. RESULTS: CD34+ cells acquired significantly more CD31 and VWF expression, increased angiogenic potential and low-density lipoprotein (ac-LDL) incorporation compared to unfractioned MNCs under the induction of VEGF. More platelets were found to adhere to the surface of unfractioned MNCs group than to the CD34+ cell group. In vivo study demonstrated that more platelet adhesion and thrombus formation were observed in the unfractioned MNCs group rather than the CD34+ group. All the grafts in both groups were patent after implantation, except one graft seeded with unfractioned MNCs, occluded at 1 week. Statistically lower ratio of thrombi was found in the CD34+ cell seeding group at 24 h and 1 week after implantation, compared with the unfractioned MNCs one (P<0.05). CONCLUSION: CD34+ cell exerted better antithrombogenic property than unfractioned MNCs after seeding onto the small caliber vessel grafts.

Preliminary studies on space charge compensation by analyzing residual argon gas ion signals.

An experimental method is related to research the space charge compensation (SCC) effect in low energy intense proton beams by analyzing residual gas (RG) ion signals. The signal curves were measured with an energy spectrometer under the RG pressure from 1.2x10(-3) to 1.6x10(-2) Pa. Most of the data showed a similar trend with our theoretical predicts. From the RG ion energy spectra the potential distribution in the beam was calculated both with and without the SCC effect. Moreover, as a preliminary result, a best compensating point is achieved for the low energy beam transport transmission of 40 KeV, 60 mA H(+) beam in Peking University.

Experimental results of an electron cyclotron resonance oxygen source and a low energy beam transport system for 1 MeV integral split ring radio frequency quadruple accelerator upgrade project.

To meet the requirements of developing separated function radio frequency quadruple (rfq) and upgrading the 1 MeV integral split ring rfq accelerator, an electron cyclotron resonance O(+) ion source and low energy beam transport (LEBT) system have been developed. Using two Einzel lenses to focus the beam, more than 6 mA O(+) peak beam current with energy of 22 keV can be easily obtained at the end of LEBT when the duty faction is at 1/6. The normalized root-mean-square emittance of 90% of the beam is about 0.12pi mm mrad. By changing the focusing power of lenses, the beam waist can be shifted from 80 mm before the beam diaphragm 2 to 80 mm after it. The experimental results will be presented in this article.

Tamoxifen metabolism in rat liver microsomes: identification of a dimeric metabolite derived from free radical intermediates by liquid chromatography/mass spectrometry.

Tamoxifen has been shown to be a potent liver carcinogen in rats, and generates covalent DNA adducts. On-line high performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been used to further study the metabolites of tamoxifen formed by rat liver microsomes in the presence of NADPH with a view to identifying potential reactive metabolites which may be responsible for the formation of DNA adducts, and liver carcinogenesis. A metabolite has been detected with a protonated molecule at m/z 773. The mass of this compound is consistent with a dimer of hydroxylated tamoxifen (m/z 388). Analysis of 4-hydroxytamoxifen incubated with a rat liver microsomal preparation showed the formation of a similar metabolite with an apparent MH+ ion at m/z 773, believed to be a dimer of 4-hydroxytamoxifen formed by a free radical reaction. The retention time for this metabolite from 4-hydroxytamoxifen is identical to that of the tamoxifen metabolite, suggesting that these two compounds are the same. The levels of the dimer were higher when 4-hydroxytamoxifen was used as substrate and, in addition, two isomers were detected. It is proposed that tamoxifen was first converted to arene oxides which react with DNA or to 4-hydroxytamoxifen, either directly or via 3,4-epoxytamoxifen, which then undergoes activation via a free radical reaction to give reactive intermediates which can then react with DNA and protein, or with themselves, to give the dimers (m/z 773).

On-line high-performance liquid chromatographic-electrospray ionization mass spectrometric method for the study of tamoxifen metabolism.

An on-line high-performance liquid chromatographic (HPLC)-electrospray ionization mass spectrometric (ESI-MS) method has been developed and optimized for the study of tamoxifen metabolism. Metabolism in mouse liver microsomes was chosen to demonstrate the applicability and superiority of the method, since mice metabolize tamoxifen faster and produce more metabolites than rats or humans. Mouse liver microsomal preparations were incubated with tamoxifen in the presence of NADPH and MgCl2. The metabolites formed were separated and analyzed by the optimized HPLC-ESI-MS system. The separation was performed on a Res Elute-BD column (5 microns particle size, 250 x 4.6 mm I.D.) with 70% (v/v) methanol in 0.5 M ammonium acetate as the mobile phase. A total of eleven metabolites have been detected, some of which have not been previously reported. The metabolites identified are: tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4'-hydroxytamoxifen, 4-hydroxytamoxifen N-oxide, 4'-hydroxytamoxifen N-oxide, 4-hydroxy-N-desmethylamoxifen, 4'-hydroxy-N-desmethyltamoxifen, 3,4-dihydroxytamoxifen, 3,4-epoxytamoxifen and 3,4-epoxytamoxifen N-oxide.

A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes.

The metabolisms of tamoxifen in female rat, mouse and human liver microsomal preparations were compared. Rat, mouse and human liver microsomes were incubated with tamoxifen in the presence of NADPH and MgCl2 and the metabolites formed were analysed by on-line HPLC-electrospray ionization MS. The major metabolites formed by rat liver microsomes were 4-hydroxytamoxifen, 4'-hydroxytamoxifen, N-desmethyltamoxifen and tamoxifen N-oxide. In addition, two epoxide metabolites, 3,4-epoxytamoxifen and 3',4'-epoxytamoxifen, and their hydrolysed derivatives, 3,4-dihydrodihydroxytamoxifen and 3',4'-dihydrodihydroxytamoxifen, have been identified. The pattern of the main metabolites obtained with human liver microsomes resembles qualitatively that of rat liver microsomes. The major differences between rat and human liver microsomes were that the amount of hydroxylated metabolites were much lower in human and only traces of 3,4-epoxytamoxifen and the corresponding dihydrodihydroxy derivative were detected. No 3',4'-epoxytamoxifen was detected in human liver microsomes. The four major metabolites were also formed in much larger amounts and with faster rates of formation by mouse liver microsomes, though tamoxifen N-oxide clearly predominated in this species. Polar metabolites, 3,4-dihydroxytamoxifen and 4-hydroxytamoxifen N-oxide, which were undetectable in rat and human, were formed in significant amounts in mouse microsomes. As in human microsomes, there was only one epoxide metabolite, 3,4-epoxytamoxifen, produced by mouse liver microsomes at levels lower than that found in rat. The faster rate of metabolism and the production of polar metabolites may indicate the ability of mouse to detoxify tamoxifen by rapid elimination compared with rat and human. The production of a larger amount of potentially reactive epoxide metabolites in rat may be responsible for the liver carcinogenesis in this species.

Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s.

The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is more genotoxic than toremifene.