[Retrospective analysis of 350 cases with dissection of lymph nodes posterior to right recurrent laryngeal nerve in endoscopic thyroidectomy through gasless axillary posterior approach].

Objective: To evaluated the safety and feasibility of dissection of lymph nodes posterior to right recurrent laryngeal nerve (ⅥB compartment) in endoscopic thyroidectomy through gasless axillary posterior approach. Methods: A total of 350 cases with right lobe papillary thyroid carcinoma (PTC) who underwent endoscopic lobectomy, isthmusectomy and central compartment neck dissection via gasless axillary posterior approach based at the Department of General Surgery, Nanfang Hospital, Southern Medical University from June 2020 to December 2022 were retrospectively analyzed. Summarize the clinical, pathological characteristics, and postoperative complications of the patients. SPSS 25.0 was used for statistical analysis of the data. Results: All 350 patients underwent endoscopic surgery successfully, with no conversion to open surgery. There were 303 females and 47 males, with an average age of (36.3±9.2) years. Of those, 287 patients were in pT1a stage, 62 in pT1b stage, and one patient in pT2 stage. There was no T3 or T4 stage patient. The mean numbers of yielded lymph nodes in right central compartment and ⅥB compartment were 8.11±4.65 (range, 1-31) and 2.62±1.86 (range, 1-12), respectively. ⅥB compartment metastasis was detected in 52 (14.86%) of 350 patients. The incidence of transient recurrent laryngeal nerve injury was 0.86%(3/350). Postoperative hematoma occurred in three patients (0.86%). Conclusion: The dissection of ⅥB compartment in endoscopic thyroidectomy through gasless axillary posterior approach is safe and feasible in selected PTC patients.

Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm.

The enhanced green fluorescent protein (EGFP) pEGFP-N1-P53 eukaryotic expression vector, which contains the human tumor suppressor p53, was constructed and transfected into chicken fibroblast cells and stage-X blastoderm to analyze the transfection efficiency. The complementary DNA of the human p53 gene was cloned by reverse transcription-polymerase chain reaction from human peripheral blood and inserted into the pEGFP-N1 vector by HindIII and BamHI double digestion. The pEGFP-N1-P53 vector was transfected into chicken embryo fibroblasts by Lipofectamine 2000 liposomes, and the transfection efficiency was analyzed by fluorescence microscope after 36 h of transfection. The stage-X blastoderm was also transfected by blastoderm injection using Lipofectamine 2000 liposomes at room temperature after 12-24 h; then hatching occurred until seventh day, and the transfection efficiency was analyzed by fluorescence microscope in the dead embryo. A total of 90 hatching eggs were transfected by the pEGFP-N1-P53 vector, and 20 chicken embryos expressed the reporter gene, which indicated that recombinant pEGFP-N1-P53 could be transfected and expressed in stage-X blastoderm by liposomes. Chicken embryo fibroblasts were transfected and expressed the reporter gene. The pEGFP-N1-P53 vector was constructed successfully and could be transfected and expressed in chicken embryo fibroblasts and stage-X blastoderms efficiently.

Immunotherapy of disseminated fibrosarcoma in mice using IL-2-producing tumor cells: studies on its mechanism and specificity.

Genetically modified, IL-2-producing tumor cells have been shown to regress in vivo and immunize mice against subsequent challenge with parental tumor. We investigated whether IL-2-producing tumor cells may serve as immunotherapy of established tumors in mice. MCA 205 and MCA 203, weakly immunogenic murine sarcomas of B6 origin, were transfected with the pBMGNeo-mIL-2 vector containing the murine IL-2 cDNA. Mice receiving intraperitoneal injections of the parental sarcoma cells developed ascites and died within 4 weeks. The intraperitoneal injection of IL-2-producing tumor cells significantly prolonged survival and, moreover, significantly reduced the number of established pulmonary metastases. The specificity of this effect was indicated by the unaltered course of disease in mice that were injected with unrelated IL-2-producing tumor cells. FACS analysis of peritoneal cells obtained from treated mice showed a predominance of Vbeta3-positive cells. In a 4 h 51Cr release assay, these Vbeta3-positive cells exhibited tumor-specific cytotoxicity and also nonspecific effector cells are shown to be involved in tumor rejection.

Phorbol ester preferentially stimulates mouse fornical conjunctival and limbal epithelial cells to proliferate in vivo.

PURPOSE: The authors investigated whether fornical epithelium displays a differential in vivo response to acute and chronic stimulation when compared with bulbar and palpebral epithelia. METHODS: To induce an increase in epithelial proliferation, 0.5% phorbol myristate (TPA) was topically applied in petrolatum daily to both eyes of SENCAR mice for 12 days. Control mice (three per group) received petrolatum only. After 6, 12, 18, and 24 hours (acute) and 2, 3, 4, 5, 7, 9, and 12 days (chronic) of TPA treatment, mice (three per group) were administered intraperitoneally 0.1 ml 40 microCi [3H]thymidine ([3H]TdR) 1 hour before they were killed. Conjunctival epithelium was fixed and processed for autoradiography, and the labeling index (LI; number of [3H]TdR-labeled nuclei per 1000 basal keratinocytes) was determined for each of the epithelial zones. RESULTS: Under normal situations, the LI was lowest in fornical epithelium (1.9 +/- 0.5) compared with bulbar (4.4 +/- 0.9) and palpebral (5.5 +/- 0.5) epithelia. Within 24 hours of TPA treatment, a 12-fold increase in fornical basal cell labeling was noted compared with a 2.5- and 5-fold increase in bulbar and palpebral basal cell labeling, respectively. Fornical epithelium maintained a significantly greater proliferative response (4.5-fold increase) during chronic stimulation than either bulbar or palpebral epithelia (0.5- and 1.5-fold increase, respectively). CONCLUSIONS: The more vigorous response of the fornical epithelium to acute and chronic stimulation is strong evidence that this epithelium has a greater proliferative capacity than the other two epithelia, which is consistent with the authors' hypothesis that conjunctival epithelial stem cells are primarily located in the fornical region.

Clonal analysis of the in vivo differentiation potential of keratinocytes.

PURPOSE: This study investigated the in vivo differentiation of conjunctival keratinocytes. METHODS: Keratinocytes from the fornical region of the conjunctival epithelium were isolated and plated at low density (5 x 102 per 100-mm dish) in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. At this density, only single, isolated cells were attached after overnight culture. Eight days later, small, well-isolated colonies separated from one another by the feeder cells were detached as a sheet from the dish and were injected subcutaneously into the flanks of BALB/c athymic mice through an 18-gauge needle. Within a day, a small firm nodule appeared at the site of injection. At different time points, the animals were killed, and the nodules were excised for morphologic, histogeometric, and cell kinetic analyses. RESULTS: Each implanted colony derived from a single cell gave rise to a single epithelial cyst lined with a reconstituted stratified epithelium. Goblet-like cells loaded with periodic acid-Schiff-positive cytoplasmic granules began to appear singularly in some of the cysts by day 8 postimplantation and were observed in approximately 85% of the cysts by day 14. CONCLUSIONS: Because the cysts formed were derived from clonal populations of epithelial cells and the majority of cysts had a mixed keratinocyte-goblet cell phenotype, these results suggest strongly the existence of a bipotent precursor cell in conjunctival epithelium that can give rise to both goblet and nongoblet cells. This system can be used to study factors that can influence the commitment of pluripotent epithelial stem cells to divergent pathways of differentiation.

Rabbit conjunctival and corneal epithelial cells belong to two separate lineages.

PURPOSE: This study investigated rabbit conjunctival and corneal epithelial cells to determine if they belong to two separate lineages. METHODS: Rabbit corneal, limbal, and conjunctival epithelial cells were isolated and grown in Dulbecco's minimum essential media and 20% fetal bovine serum in the presence of mitomycin-treated 3T3 feeder cells. After reaching 80% confluence, 3T3 feeder cells and any contaminating fibroblasts were removed, and epithelial cells were resuspended in fresh Dulbecco's minimum essential media. Aliquots containing 5x10(6) cells were placed subcutaneously into the flanks of athymic mice, which subsequently formed small nodules. At 2, 4, 6, 8, 14, 21, and 28 days, athymic mice were killed and the nodules (epithelial cyst) were excised for light and transmission electron microscopic examination and histochemical and cell kinetic analyses. RESULTS: Within 2 days after injection of single-cell suspensions, cells aggregated to form cysts lined with a stratified squamous epithelium, the structure of which resembled the original in vivo donor sites by 8 days. Limbal- and corneal-derived cysts were comprised only of glycogen-rich stratified epithelial cells. In contrast, only cysts arising from cultured conjunctival cells contained periodic acid-Schiff-positive cells with a goblet cell structure interspersed among stratified epithelial cells. Furthermore, cystic epithelium of conjunctival origin did not accumulate glycogen. CONCLUSIONS: To determine whether distinct phenotypes are caused by intrinsic divergence or by environmental modulation, the behavior of cells can be monitored in an identical in vivo growth environment. The athymic mouse provides such a permissive growth environment for cultured corneal, limbal, and conjunctival epithelial cells. All these cells reproduced their in vivo phenotype when placed in the athymic mouse. Thus, these findings provide the strongest evidence to date that the corneal-limbal lineage is distinct from the conjunctival lineage. These data also support the idea that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment.

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis.

PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.

Lineage-specific and differentiation-dependent expression of K12 keratin in rabbit corneal/limbal epithelial cells: cDNA cloning and northern blot analysis.

Corneal epithelial cells synthesize an acidic (55 kDa) K12 and a basic (64 kDa) K3 keratin as their major differentiation products during an advanced stage of differentiation. In this paper, we describe the cDNA cloning of rabbit K12 keratin. We used a 36 base pairs (bp) oligonucleotide corresponding to a consensus sequence of many known acidic keratins as a probe to screen a cDNA library of normal rabbit corneal epithelium. Several partial cDNA clones were isolated. Hybrid-selection showed that the 3'keratin chain-specific portion of the cDNA hybridizes with K12 mRNA. A rabbit antiserum raised against the C-terminus of the cDNA-deduced amino acid sequence recognizes, in immunoblotting, the K12 keratin. In situ hybridization showed that K12 mRNA is present in all cell layers of central corneal epithelium, but in only the suprabasal cells of limbal epithelium indicating a parallel expression pattern between K12 and K3. Cultured rabbit corneal epithelial cells initially synthesize K14/K5 keratins, but later when the cells become heavily stratified they synthesize large quantities of K12 and K3 mRNAs, as detected by Northern blotting. Cultured esophageal epithelial cells do not make K12 mRNA confirming the tissue-specificity of K12 expression. Although it has been suggested that conjunctival epithelial cells can trans-differentiate into a bona fide corneal epithelium, we showed here that cultured conjunctival cells do not synthesize significant amounts of K12/K3 mRNAs. These results strongly suggest that conjunctival epithelial cells, whose differentiation can be modulated significantly by the extracellular matrix, form a lineage intrinsically distinct from the corneal/limbal epithelial lineage.

Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: possible involvement of hair follicle stem cells.

Stem cells are believed to be a necessary target of chemical carcinogens. Based on autoradiographic, ultrastructural, and biologic criteria, we have recently proposed that hair follicle stem cells reside not in the bulb, but in the upper outer root sheath in an area called the bulge. Proliferating cells have been shown to be more susceptible to tumor initiation, and we have recently demonstrated that cells in the bulge undergo transient proliferation during early anagen. Therefore, we theorized that mouse skin should be particularly susceptible to carcinogen application during early anagen phase. In this paper, we show that early anagen Swiss and Sencar mouse skin is indeed particularly susceptible to one- and two-stage chemical carcinogenesis, resulting in tumor yields one to five times those obtained with telogen-timed carcinogen application. Our findings implicate a possible involvement of the bulge cells as precursors to some of the skin cancers, and support the concept that these are stem cells. These observations also raise important questions about the cellular origins and biologic behavior of chemically induced murine skin tumors.

In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells.

PURPOSE: The anterior surface of the eye is covered by several physically contiguous but histologically distinguishable epithelial overlying the cornea, limbus, bulbar conjunctiva, fornix conjunctiva, and palpebral conjunctiva. It is important to determine whether the different phenotypes of these epithelia are the result of intrinsic divergence, extrinsic modulation, or a combination of both. Based on keratin expression and cell kinetic criteria, the authors previously suggested that corneal epithelial stem cells may actually reside in the limbal basal layer. METHODS: In this article, the relationship between the corneal-limbal epithelial cells and conjunctival epithelial cells was analyzed by comparing their growth and differentiation properties in an identical cell culture environment. RESULTS: Using Dispase instead of trypsin to dissociate the cells, the authors were able to grow all five rabbit ocular surface epithelia in the presence of 3T3 feeder cells. They found that corneal and limbal cells synthesize identical keratins, including large amounts of the K3 and K12 markers of corneal-type differentiation. By contrast, all three conjunctival epithelia shared another keratin pattern, with large amounts of simple epithelial keratins but only minute amounts of K3/K12 keratins. CONCLUSIONS: This observation, coupled with previous findings that the "transdifferentiation" of conjunctival epithelial cells to corneal epithelium appears to be both incomplete and reversible, provides strong evidence that (1) the limbal-corneal epithelial cells form a lineage distinct from the conjunctival lineage and (2) conjunctival transdifferentiation actually represents a process of environmental modulation. In addition, of the three types of conjunctival epithelial cells, fornix cells were found to have a much greater proliferative potential than bulbar and palpebral cells. This observation, coupled with recent finding that fornix is enriched in slow-cycling (label-retaining) cells, raises the possibility that conjunctival epithelial stem cells may preferentially reside in the fornix.